Amino acid modifications in canine, equine and porcine pituitary growth hormones, identified by peptide-mass mapping

Modified amino acid residues in porcine, canine and equine growth hormones purified from pituitary glands were characterised by tryptic mapping and high-performance liquid chromatography with on-line coupled electrospray ionisation mass spectrometry (HPLC–ESI-MS) detection. Hormones from all three species showed the same changes. Conversion of Asp¹²⁸ to iso-Asp¹²⁸ was a component of native hormones, while deamidation of Asn¹² and Asn⁹⁸ to Asp and iso-Asp, oxidation of Met⁴, and cyclisation to the pyroglutamyl derivative of Gln¹³⁹, probably occurred in vitro, during isolation, storage or hydrolysis. Porcine and canine hormones had indistinguishable protein fingerprints, confirming the assumption, based on their cDNA sequences, that their mature primary structures are identical.     

Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells

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Global distribution and status of introduced Siberian chipmunks Eutamias sibiricus

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Alteration of bacterial communities and organic matter in microbial fuel cells (MFCs) supplied with soil and organic fertilizer

The alteration of the organic matter (OM) and the composition of bacterial community in microbial fuel cells (MFCs) supplied with soil (S) and a composted organic fertilizer (A) was examined at the beginning and at the end of 3 weeks of incubation under current-producing as well as no-current-producing conditions. Denaturing gradient gel electrophoresis revealed a significant alteration of the microbial community structure in MFCs generating electricity as compared with no-current-producing MFCs. The genetic diversity of cultivable bacterial communities was assessed by random amplified polymorphic DNA (RAPD) analysis of 106 bacterial isolates obtained by using both generic and elective media. Sequencing of the 16S rRNA genes of the more representative RAPD groups indicated that over 50.4% of the isolates from MFCs fed with S were Proteobacteria, 25.1% Firmicutes, and 24.5% Actinobacteria, whereas in MFCs supplied with A 100% of the dominant species belonged to γ-Proteobacteria. The chemical analysis performed by fractioning the OM and using thermal analysis showed that the amount of total organic carbon contained in the soluble phase of the electrochemically active chambers significantly decreased as compared to the no-current-producing systems, whereas the OM of the solid phase became more humified and aromatic along with electricity generation, suggesting a significant stimulation of a humification process of the OM. These findings demonstrated that electroactive bacteria are commonly present in aerobic organic substrates such as soil or a fertilizer and that MFCs could represent a powerful tool for exploring the mineralization and humification processes of the soil OM.     

FAD binding in glycine oxidase from Bacillus subtilis

The apoprotein of the FAD-containing flavoenzyme glycine oxidase from Bacillus subtilis was obtained at pH 8.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M Tris–HCl and 20% glycerol. The apoprotein of glycine oxidase shows high protein fluorescence, high exposure of hydrophobic surfaces, and low temperature stability as compared to the holoenzyme. The isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD. The reconstitution process follows a particular two-stage process; the spectral properties of the reconstituted holoenzyme were virtually indistinguishable from those observed with native glycine oxidase, while the activity was only partially (50%) recovered. The urea-induced unfolding process of glycine oxidase can be considered as a two-step (three-state) process: the presence of intermediate(s) in the unfolding process of the holoenzyme at ≈2 M urea is evident in the changes of the flavin fluorescence intensity and can be also inferred from the different urea sensitivities of the spectral probes used. On the other hand, only a single transition at ≈4.5 M urea concentration is observed for the apoprotein form. The chemical denaturation of glycine oxidase holoenzyme is partially reversible (e.g., no activity is recovered when starting the refolding from 4 M urea-denatured holoprotein). Finally, the introduction by site-directed mutagenesis of residues corresponding to those involved in the covalent link with FAD in the related flavoenzyme monomeric sarcosine oxidase failed to convert glycine oxidase into a covalent flavoprotein. These investigations show that the consequences of FAD binding for the stability and folding process distinguish glycine oxidase from enzymes active on similar compounds.     

What are we dealing with? An explicit test reveals different levels of taxonomical diagnosability in the Sylvia cantillans species complex

Diagnosability is the ability to discriminate between similar taxa, including sibling or cryptic taxa. We have developed an explicit test of diagnosability, using the Sylvia cantillans species complex as a model, which compares an identification based on phenotype with that based on genotype. Individual warblers sampled during their migration in central Italy were first identified to the (sub)species level using putatively diagnostic plumage traits. Nucleotide sequences of a (598-bp) fragment of the mitochondrial (mt)DNA cytochrome b were then used to assign each individual to distinct phylogenetic clades, as determined by reference haplotypes that had been sequenced in breeding individuals. This resulted in the construction of clearly distinct clades corresponding to known taxa of the complex. The new haplotypes were assigned to one of the previously identified groups (corresponding to three different taxa); no sample was assigned outside of them. In contrast, when plumage traits were used to assign the birds into distinct phylogenetic clades, 11 of 58 birds were classified as ‘uncertain/intermediate’ among two taxa, while five were classified differently with the two methods. A perfect agreement between the two methods was found for only for one taxon (Sylvia subalpina, syn. S. moltonii). For the other two taxa of the complex, diagnosability is therefore not guaranteed, and their field identification by hand should be carefully addressed. We provide here an example of an explicit test for establishing the diagnosability of taxa in which two or more ‘markers’ can be used for determining discordant identification and/or unambiguous diagnosability. Our results outline the importance of considering different features for taxa diagnosis and illustrate the weakness of visual appearance-based identification (currently widely used for taxa determination) in our study complex.