Crystallization and preliminary crystallographic data of the PAS domain of the NifL protein from Azotobacter vinelandii.

The Azotobacter vinelandii NifL protein is a redox-sensing flavoprotein which inhibits the activity of the nitrogen-specific transcriptional activator NifA. The N-terminal PAS domain has been overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method. The crystal belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 65.0, c = 157.3 Å, and has one molecule in the asymmetric unit. Native data were collected to 3.0 Å on the BW7B synchrotron beamline at the EMBL Hamburg Outstation

Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.

Five glucosyltransferases were cloned by RT-PCR amplification using total RNA from Hieracium pilosella L. (Asteraceae) inflorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially purified and functionally characterised as UDP-glucose:flavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed different regiospecificity profiles. UGT95A1 established a new subgroup within the UGT family showing high regiospecificity towards the C-3' hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4'-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this flavones glucoside in H. pilosella flower heads. Semi quantitative expression profiles revealed that UGT95A1 was expressed at all stages of inflorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to flower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards flavones and flavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolite

Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance

Nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS) are frequently used as technological platforms for metabolomics applications. In this study, the metabolic profiles of ripe fruits from 50 different tomato cultivars, including beef, cherry and round types, were recorded by both 1H NMR and accurate mass LC-quadrupole time-of-flight (QTOF) MS. Different analytical selectivities were found for these both profiling techniques. In fact, NMR and LCMS provided complementary data, as the metabolites detected belong to essentially different metabolic pathways. Yet, upon unsupervised multivariate analysis, both NMR and LCMS datasets revealed a clear segregation of, on the one hand, the cherry tomatoes and, on the other hand, the beef and round tomatoes. Intra-method (NMR¿NMR, LCMS¿LCMS) and inter-method (NMR¿LCMS) correlation analyses were performed enabling the annotation of metabolites from highly correlating metabolite signals. Signals belonging to the same metabolite or to chemically related metabolites are among the highest correlations found. Inter-method correlation analysis produced highly informative and complementary information for the identification of metabolites, even in de case of low abundant NMR signals. The applied approach appears to be a promising strategy in extending the analytical capacities of these metabolomics techniques with regard to the discovery and identification of biomarkers and yet unknown metabolites

Proteomic analysis of Glossina pallidipes salivary gland hypertrophy virus virions for immune intervention in tsetse fly colonies

Many species of tsetse flies (Diptera: Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH). The viruses isolated from Glossina pallidipes (GpSGHV) and Musca somestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have a reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programs to control and eradicate tsetse populations in the wild. A potential intervention strategy to mitigate viral infections in fly colonies is neutralizing of the GpSGHV infection with specific antibodies against virion proteins. Two major GpSGHV virion proteins of about 130 kDa and 50 kDa, respectively, were identified by Western analysis using polyclonal rabbit antibody raised against whole GpSHGV virions. The proteome of GpSGHV, containing the antigens responsible for the immune-response, was investigated by liquid chromatography tandem mass spectrometry (LC-MS/MS) and 61 virion proteins were identified by comparison with the genome sequence. Specific antibodies were produced in rabbits against seven candidate proteins including the ORF10 / C-terminal fragment, ORF47 and ORF96 as well as proteins involved in peroral infectivity PIF-1 (ORF102), PIF-2 (ORF53), PIF-3 (ORF76) and P74 (ORF1). Antiserum against ORF10 specifically reacted to the 130 kDa protein in a Western blot analysis and to the envelope of GpSGHV using immunogold-EM. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic optio

Chemical Identification Strategies Using Liquid Chromatography-Photodiode Array-Solid-Phase Extraction-Nuclear Magnetic Resonance/Mass Spectroscopy.

The identification of metabolites in biochemical studies is a major bottleneck in the proliferating field of metabolomics. In particular in plant metabolomics, given the diversity and abundance of endogenous secondary metabolites in plants, the identification of these is not only challenging but also essential to understanding their biological role in the plant, and their value to quality and nutritional attributes as food crops. With the new generation of analytical technologies, in which liquid chromatography (LC)-mass spectrometry (MS) and nuclear magnetic resonance (NMR) play a pioneering role, profiling metabolites in complex extracts is feasible at high throughput. However, the identification of key metabolites remains a limitation given the analytical effort necessary for traditional structural elucidation strategies. The hyphenation of LC-solid phase extraction (SPE)-NMR is a powerful analytical platform for isolating and concentrating metabolites for unequivocal identification by NMR measurements. The combination with LC-MS is a relatively straightforward approach to obtaining all necessary information for structural elucidation. Using this set-up, we could, as an example, readily identify five related glycosylated phenolic acids present in broccoli (Brassica oleracea, group Italica, cv Monaco): 1,2-di-O-E-sinapoyl-ß-gentiobiose, 1-O-E-sinapoyl-2-O-E-feruloyl-ß-gentiobiose, 1,2-di-O-E-feruloyl-ß-gentiobiose, 1,2,2'-tri-O-E-sinapoyl-ß-gentiobiose, and 1,2'-di-O-E-sinapoyl-2-O-E-feruloyl-ß-gentiobiose

In vitro digestibility and fermentability of selected insects for dog foods

Insects are considered as a sustainable protein source for future pet foods. Here we aimed to evaluate the protein quality of larvae of the black soldier fly (Hermetia illucens, BSF), housefly (Musca domestica, HF) and yellow mealworm (Tenebrio molitor, YMW) and to evaluate the fermentation characteristics of their indigestible fractions. Clean freeze-dried larvae were subjected to in vitro simulated canine gastric and small intestinal digestion. Undigested insect residues, shrimp chitin and fructooligosaccharides (positive control, FOS) were incubated for 48 h with inoculum with fresh feces from three dogs simulating large intestinal fermentation. The AA profiles differed among the larvae with proteins from BSF and YMW larvae containing more Val and less Met and Lys than HF larvae. The in vitro N digestibility of the HF (93.3%) and YMW (92.5%) was higher than BSF larvae (87.7%). The BSF larvae also had lower in vitro digestibility values for essential AA (92.4%) and non-essential AA (90.5%) compared to the larvae of the HF (96.6 and 96.5%) and YMW (96.9 and 95.3%). Gas production for FOS increased rapidly during the first 6 h. Low and similar amounts of gas were found for HF larvae and chitin whereas gas production slowly increased over 30 h and was slightly higher at 48 h for BSF than for chitin. Gas production for YMW increased considerably between 6 and 20 h. At 48 h, gas produced for undigested residues was comparable to shrimp chitin and lower than FOS (P </p

Plant Micrometabolomics: The Analysis of Endogenous Metabolites Present in a Plant Cell or Tissue.

Identification and quantification of metabolites occurring within specific cell types or single cells of plants and other organisms is of particular interest for natural product chemistry, chemical ecology, and biochemistry in general. The integration of studies at the gene, transcript, protein and metabolite levels in localized regions will provide useful information for the understanding of biology as a system. In this review, we summarize the latest developments in the analysis of metabolites present in small samples, micrometabolomics, dealing with sample preparation methods, with focus on laser-assisted microdissection, and the analytical technologies used. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) are among the most emergent technologies in metabolomics, enabling the shortest route toward metabolite identification