Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.

Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCKrelated proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs.Receptor protein kinases (RPKs) 1 are essential components of the cell regulation machinery that transmits extracellular signals to the inside of the cell. Although RPKs show high variability in their receptor domain, they share highly conserved cytoplasmic kinase domains, and they are assumed to function by a relatively well conserved general mechanism. After ligand binding and receptor oligomerization, the intracellular kinase domain becomes activated. This results in intermolecular auto-phosphorylation and conformational changes that allow the receptor to bind downstream signaling proteins (see Refs. 1 and 2). Nevertheless, atypical RPKs that transduce signals by phosphorylation-independent mechanisms have been described recently (3). They include phosphorylation-capable PRKs that can also signal through other mechanisms but also completely kinase-defective atypical RPKs, such as CCK-4 (4), H-Ryk (5, 6), 8), and DNT (9). These atypical RPKs have substitutions within the kinase-conserved motifs, especially in the aspartic acid in subdomain VIb and in the DFG activation loop motif of subdomain VII. Among them, ErbB3 and H-Ryk are probably the best known examples. It has been shown that mice knockouts for ErbB-3 or Ryk present severe mutant phenotypes suggesting that both proteins are essential for signaling (10, 11). ErbB3 forms heterodimers with other members of the family of epidermal growth family receptors and is phosphorylated by these kinase-active RPKs (12). Upon phosphorylation, ErbB3 serves as docking sites for multiple downstream signaling proteins. H-Ryk also forms heterodimers with other kinase-active RPKs, although in this case the interaction does not result in phosphorylation (13). A chimeric receptor approach has shown that the ligand stimulation of H-Ryk results in activation of the MAPK pathway (6), suggesting that activated H-Ryk can interact with and activate other downstream signaling proteins. Indeed, it has been proposed that signaling through atypical RPKs could involve regulated protein-protein interactions through their intracellular domains, which wo...

The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter β-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases.