The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S 50 s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 g/ml and 0.29 g/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley ␣-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (ϳ0.05 g in 50 mg of leaves) were far lower than the S 50 determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.
Purpose-Cynarin, a potential immunosuppressant that blocks the interaction between the CD28 of T-cell receptor and CD80 of antigen presenting cells, was found in Echinacea purpurea by a new pharmaceutical screening method: After Flowing Through Immobilized Receptor (AFTIR; Dong et al., J Med Chem, 49: 1845-1854, 2006. This Echinacea component is the first small molecule that is able to specifically block "signal 2" of T-cell activation.Methods-In this study, we used the AFTIR method to further confirm that cynarin effectively blocked the binding between CD80 of B-cells and CD28 of T-cells, and provide details of its mechanism of action.Results-The experimental results showed that cynarin blocked about 87% of the CD28-dependent "signal 2" pathway of T-cell activation under the condition of one to one ratio of T-cell and B-cell in vitro. Theoretical structure modeling showed that cynarin binds to the "G-pocket" of CD28 (Evans et al., Nat Immunol, 6:271-279, 2005), and thus interrupts the site of interaction between CD28 and CD80.Conclusions-These results confirm both that AFTIR is a promising method for screening selective active compounds from herbal medicine and that cynarin has great potential as an immunosuppressive agent.
The basal mitochondrial transcription apparatus of Saccharomyces cerevisiae consists of the core enzyme for mitochondrial RNA polymerase and the specificity factor. The core enzyme is homologous to those of bacteriophages T3, T7 and SP6 whereas the specificity factor shows similarities with bacterial sigma factors. Recently it was shown that the bacteriophage-type core enzyme is widespread among the eukaryotic lineage and a common picture for the mitochondrial transcription apparatus in eukaryotic cells is now emerging. In contrast to the situation for the core enzyme, the gene for the specificity factor has only been identified from S. cerevisiae and more recently from two other yeast species. As the specificity factor is the key component for initiation of transcription at the mitochondrial promoter we wanted to study in more detail gene expression, regulation, and the function of the promoter of the nuclear MTF1 gene. For this purpose the messenger RNA level for scMTF1 was investigated under a large number of different growth conditions and thereby exhibited a very low, but regulated and carbon source-dependent, expression. Deletion experiments identify the minimal promoter for functional complementation in yeast. To evaluate the functional conservation of the promoter elements the homologous MTF1 gene from the closely related yeast Saccharomyces douglasii was isolated and tested in heterologous complementation experiments. In spite of a highly conserved protein sequence these studies demonstrate that at low-copy number sdMTF1 is not able to substitute for scMTF1 in S. cerevisiae. Promoter exchange experiments with MTF1 from S. cerevisiae and S. douglasii demonstrate that differences in gene expression are responsible for the failure in heterologous complementation. This finding prompted us to compare the promoter regions of MTF1 from four different yeast species. For this purpose the sequences of the 5' regions from S. douglasii, S. kluyveri and Kluyveromyces lactis were determined. A comparison of these sequences identifies significant differences and rapid changes in the intergenic regions, even between closely related yeast species.
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