Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

Identification of (η6-arene)ruthenium(II) protein binding sites in E. coli cells by combined multidimensional liquid chromatography and ESI tandem mass spectrometry: specific binding of [(η6- p -cymene)RuCl2(DMSO)] to stress-regulated proteins and to helicases

An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(η⁶-p-cymene)RuCl₂(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (η⁶-p-cymene)Ru^II fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(η⁶-p-cymene)RuCl₂(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA repair profiles of disease-associated isolates of Neisseria meningitidis

Neisseria meningitidis, or the meningococcus, is the source of significant morbidity and mortality in humans worldwide. Even though mutability has been linked to the occurrence of outbreaks of epidemic disease, meningococcal DNA repair pathways are poorly delineated. For the first time, a collection of meningococcal disease-associated isolates has been demonstrated to express constitutively the DNA glycosylases MutY and Fpg in vivo. DNA sequence analysis showed considerable variability in the deduced amino acid sequences of MutS and Fpg, while MutY and RecA were highly conserved. Interestingly, multi-locus sequence typing demonstrated a putative link between the pattern of amino acid substitutions and levels of spontaneous mutagenicity in meningococcal strains. These results provide a basis for further studies aimed at resolving the genotype/phenotype relationships of meningococcal genome variability and mutator activity.