Lipid-to-gibberellin metabolic switching in Fusarium moniliforme via the action of sesamol

Sesamol (3,4-methylenedioxyphenol) at 2.5 mM inhibited growth of Fusarium moniliforme by about 40% and lipid accumulation by 35%. Gibberellin (GA₃) accumulation was increased by 20-fold, to 63 mg g^–1 biomass, in the presence of sesamol indicating that the acetyl-CoA destined for fatty acid biosynthesis was now being switched into secondary metabolite (GA₃) accumulation. Synthesis of other metabolites from acetyl-CoA, such as bikaverin and carotenoids, though were not increased in the presence of sesamol. Metabolic switching is therefore feasible by judicious use of selected inhibitors that can thus block primary metabolic routes but which do not affect secondary metabolites.     

Structural characterization of human elastin derived peptides containing the GXXP sequence

The degradation of elastin, the insoluble biopolymer of tropoelastin, can lead to the production of small peptides. These elastin-derived peptides (EDPs) are playing a key role in cellular behavior within the extracellular matrix, showing a great variety of biological effects such as chemotaxis, stimulation of cell proliferation, ion flux modifications, vasorelaxation, and inflammatory enzymes secretion. It has also been demonstrated recently that EDPs containing the GXXPG motif could induce pro-MMP1 and pro-MMP3 upregulation. Elastolysis could then cause collagen degradation and play an important role in the aging process. Many experimental studies have been devoted to EDPs, but their structure/activity relationships are not well elucidated yet. However, the assumption that their active conformation is a type VIII beta-turn on GXXP was highly suggested on the basis of predictive statistical calculations. Investigation of the EDPs three-dimensional (3D) structure would provide useful information for drug-design strategies to propose specific inhibitors. The work presented here reports theoretical results obtained from molecular dynamics simulations performed over 128 human EDPs containing the GXXP motif. We show that all the peptides, for which the central residues are not glycines, adopt a canonical (or very close to) type VIII beta-turn structure on the GXXP sequence. Amino acids surrounding this motif are also important for the structural behavior. Any residue located before the GXXP motif (XGXXP) increases the beta-turn stabilization, whereas the residue located after GXXP (GXXPX) has no significant structural effect. Moreover, we show their biological activity can be correlated with their ability to exhibit a type VIII beta-turn conformation.     

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.     

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.     

The antitumor properties of the alpha3(IV)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.     

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T₀, T₁, and T₂ generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T₁ and T₂ generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.