Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species

Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.     

In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid

Efficient induction of mucosal immunity usually employs nasal or oral vaccination while parenteral immunization generally is ineffective at generating mucosal immune responses. This relates to the unique ability of resident mucosal dendritic cells (DC) to induce IgA switching and to imprint mucosa-specific homing receptors on lymphocytes. Based on the well-established plasticity of the DC system, this study sought to investigate whether peripheral DC could be modulated toward "mucosa-type" DC by treatment with immunomodulatory, and therefore potentially adjuvant-like, factors. In this study, we show that monocyte-derived DCs pretreated with the vitamin A derivative all-trans retinoic acid (RA) indeed acquired several attributes characteristic of mucosal DC: secretion of TGF-beta and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in cocultured lymphocytes. Addition of a TGF-beta-neutralizing Ab to cocultures significantly inhibited alpha4beta7 integrin, but not CCR9 mRNA expression by the lymphocytes. Both alpha4beta7 integrin and CCR9 mRNA expression, but not IgA production, were suppressed in the presence of a RA receptor antagonist. None of the observed effects on the lymphocytes were influenced by citral, a retinal dehydrogenase inhibitor, arguing against a role for de novo-synthesized RA. Collectively, our findings identified a novel role for RA as a mucosal immune modulator targeting DC. Our results further demonstrate that DC can act as efficient carriers of RA at least in vitro. Consequently, RA targeting of DC shows potential for promoting vaccine-induced mucosal immune responses via a parenteral route of immunization.     

The crystal structure of Trypanosoma cruzi glucokinase reveals features determining oligomerization and anomer specificity of hexose-phosphorylating enzymes

Glucose is an essential substrate for Trypanosoma cruzi, the protozoan organism responsible for Chagas' disease. The glucose is intracellularly phosphorylated to glucose 6-phosphate. Previously, a hexokinase responsible for this phosphorylation has been characterized. Recently, we identified an ATP-dependent glucokinase in T. cruzi exhibiting a tenfold lower substrate affinity compared to the hexokinase. Both enzymes, which belong to very different groups of the same family, are located inside glycosomes, the peroxisome-like organelles of Kinetoplastida that are known to contain the first seven glycolytic steps as well as enzymes of the oxidative branch of the pentose phosphate pathway. Here, we present the crystallographic structure of T. cruzi glucokinase, in complex with glucose and ADP. The structure suggests a loose tetrameric assembly formed by the association of two tight dimers. TcGlcK was previously reported to exist in a concentration-dependent equilibrium of monomeric and dimeric states. Here, we used mass spectrometry analysis to confirm the existence of TcGlcK monomeric and dimeric states. The analysis of subunit interactions and comparison with the bacterial glucokinases give insights into the forces promoting the stability of the different oligomeric states. Each T. cruzi glucokinase monomer contains one glucose and one ADP molecule. In contrast to hexokinases, which show a moderate preference for the alpha anomer of glucose, the electron density clearly shows the d-glucose bound in the beta configuration in the T.cruzi glucokinase. Kinetic assays with alpha and beta-d-glucose further confirm a moderate preference of the T. cruzi glucokinase for the beta anomer. Structural comparison of the glucokinase and hexokinases permits the identification of a possible mechanism for anomer selectivity in these hexose-phosphorylating enzymes. The preference for distinct anomers suggests that in T. cruzi hexokinase and glucokinase are not directly competing for the same substrate and are probably both present because they exert distinct physiological functions.     

Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species

2-Styrylchromones are a small group of naturally occurring chromones, vinylogues of flavones (2-phenylchromones). Natural and synthetic 2-styrylchromones have been tested in different biological systems, showing activities with potential therapeutic applications. In particular, the potential and hitherto understudied antioxidant behavior of these compounds has been raised as a matter of interest. Thus the present work consisted in the study of the in vitro scavenging activities for reactive oxygen species (ROS) and reactive nitrogen species (RNS) of various 2-styrylchromone derivatives and structurally similar flavonoids. Some of the studied 2-styrylchromones proved to be extremely efficient scavengers of the different ROS and RNS, showing, in some cases, IC(50)s under 1 microM. The hydroxylation pattern of 2-styrylchromones, especially in the B-ring but also in the A ring, modulates the activity of these compounds, the catecholic derivatives being the most effective scavengers. The styryl pattern also contributes to their observed outstanding antioxidant activity. In conclusion, the scavenging activities for ROS/RNS of 2-styrylchromone derivatives, here shown for the first time, provide novel and most promising compounds to be applied as antioxidants.     

Family of Tc1-like elements from fish genomes and horizontal transfer

The involvement of horizontal transfer (HT) in the evolution of vertebrate transposable elements (TEs) is a matter of an ongoing debate. The phylogenetic relationships between Tc1 TEs, based on limited dataset have been previously used to infer a case of Tc1 HT between the genomes of fish and frogs. Here this hypothesis has been critically evaluated by the experimental approach including comparative data on the range of fish species available today. The distribution of a Tc1 subfamily of TE in selected fish species was investigated by PCR with a single primer complementary to ITRs and showed that they are widespread in the studied 17 fish species. They belong to five different subfamilies of Tc1 TEs, as revealed by the comparison with current genomic data for fish and amphibians. The original hypothesis would get much weaker support from the current data, although at least one novel potential and more convincing case of HT was identified between genomes of Perciformes fish. An interesting case of recombination-driven mobilisation of a degenerated TE by distantly related TE from different subfamily was discovered in the genome of pike. The occurrence of such cases widens the range of TE elements identifiable with the employed experimental approach. Further similar studies would help to explain the evolution of the multiple Tc1 lineages including species for which full genome sequences will not be available soon.     

5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites

In the human malaria parasite Plasmodium falciparum antigenic variation facilitates long-term chronic infection of the host. This is achieved by sequential expression of a single member of the 60-member var family. Here we show that the 5' flanking region nucleates epigenetic events strongly linked to the maintenance of mono-allelic var gene expression pattern during parasite proliferation. Tri- and dimethylation of histone H3 lysine 4 peak in the 5' upstream region of transcribed var and during the poised state (non-transcribed phase of var genes during the 48 h asexual life cycle), 'bookmarking' this member for re-activation at the onset of the next cycle. Histone H3 lysine 9 trimethylation acts as an antagonist to lysine 4 methylation to establish stably silent var gene states along the 5' flanking and coding region. Furthermore, we show that competition exists between H3K9 methylation and H3K9 acetylation in the 5' flanking region and that these marks contribute epigenetically to repressing or activating var gene expression. Our work points to a pivotal role of the histone methyl mark writing and reading machinery in the phenotypic inheritance of virulence traits in the malaria parasite.