Cage bedding modifies metabolic and gut microbiota profiles in mouse studies applying dietary restriction.

Experiments involving food restriction are common practice in metabolic research. Under fasted conditions, mice supplement their diet with cage bedding. We aimed at identifying metabolic and microbiota-related parameters affected by the bedding type. We exposed mice housed with wooden, cellulose, or corncob cage beddings to ad libitum feeding, caloric restriction (CR), or over-night (ON) fasting. Additionally, two subgroups of the ON fast group were kept without any bedding or on a metal grid preventing coprophagy. Mice under CR supplemented their diet substantially with bedding; however, the amount varied depending on the kind of bedding. Bedding-related changes in body weight loss, fat loss, cecum size, stomach weight, fecal output, blood ghrelin levels as well as a response to glucose oral tolerance test were recorded. As fiber is fermented by the gut bacteria, the type of bedding affects gut bacteria and fecal metabolites composition of CR mice. CR wood and cellulose groups showed distinct cecal metabolite and microbiome profiles when compared to the CR corncob group. While all ad libitum fed animal groups share similar profiles. We show that restriction-related additional intake of bedding-derived fiber modulates multiple physiological parameters. Therefore, the previous rodent studies on CR, report the combined effect of CR and increased fiber consumption.

Identification of putative new tomato allergens and differential interaction with IgEs of tomato allergic subjects.

BACKGROUND: Tomato became one of the world-wide most consumed vegetables, unfortunately accompanied by an increasing risk of tomato allergy affecting certain people. As tomato allergic subjects show highly variable reactions in clinical allergy tests, it is difficult to identify cultivars or differentially treated tomato plants where a significant reduction in the allergenic potential over all subjects of a cohort can be detected. OBJECTIVE: This study was carried out to test the hypothesis that individual variability is based on differential reactions of single subjects to particular allergens in tomato fruits of plants with certain genetic background or cultivated under distinct conditions. METHODS: Proteins were extracted from tomato fruits of the previously investigated genotypes 76R, its mycorrhizal mutant RMC, and the cultivar Counter, fertilized with different forms of nitrogen in deficit or excess. 2-D immunoblots were carried out with sera of nine tomato allergic subjects, beforehand analysed in skin prick tests. RESULTS: In total, ten putative tomato allergens were identified in these immunoblots. No correlation was detected between individual skin prick test results and the quantity of positive reactions to putative allergens. IgEs of each subject showed reactions to nearly every identified putative allergen, but reactions were dependent on genotype and growth conditions. Among the ten putative tomato allergens, five new candidates were identified as follows: an endo-ß-mannanase, a pectinacetylesterase, a pectinesterase inhibitor, an aspartyl protease family protein and a protein of unknown function. CONCLUSION AND CLINICAL RELEVANCE: The hypothesis that high interindividual differences in allergic reactions are based on the interactions between the IgEs of allergic subjects with particular allergens has to be rejected. However, five proteins with putative clinical relevance as tomato allergens could be newly identified.

Metabolomic networks in plants: Transitions from pattern recognition to biological interpretation.

Nowadays techniques for non-targeted metabolite profiling allow for the generation of huge amounts of relevant data essential for the construction of dynamic metabolomic networks. Thus, metabolomics, besides transcriptomics or proteomics, provides a major tool for the characterization of postgenomic processes. In this work, we introduce comparative correlation analysis as a complementary approach to characterize the physiological states of various organs of diverse plant species with focus on specific participation of metabolites in different reaction networks. The correlations observed are induced by diminutive fluctuations in environmental conditions, which propagate through the system and induce specific patterns depending on the genomic background. In order to examine this hypothesis, numeric examples of such fluctuations are computed and compared with experimentally obtained metabolite data.

Interpreting correlations in metabolomic networks.

Correlations, as observed between the concentrations of metabolites in a biological sample, may be used to gain additional information about the physiological state of a given tissue. In this mini-review, we discuss the integration of these observed correlations into metabolomic networks and their relationships with the underlying biochemical pathways.

Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).

Observing and interpreting correlations in metabolomic networks.

MOTIVATION: Metabolite profiling aims at an unbiased identification and quantification of all the metabolites present in a biological sample. Based on their pair-wise correlations, the data obtained from metabolomic experiments are organized into metabolic correlation networks and the key challenge is to deduce unknown pathways based on the observed correlations. However, the data generated is fundamentally different from traditional biological measurements and thus the analysis is often restricted to rather pragmatic approaches, such as data mining tools, to discriminate between different metabolic phenotypes. METHODS AND RESULTS: We investigate to what extent the data generated networks reflect the structure of the underlying biochemical pathways. The purpose of this work is 2-fold: Based on the theory of stochastic systems, we first introduce a framework which shows that the emergent correlations can be interpreted as a 'fingerprint' of the underlying biophysical system. This result leads to a systematic relationship between observed correlation networks and the underlying biochemical pathways. In a second step, we investigate to what extent our result is applicable to the problem of reverse engineering, i.e. to recover the underlying enzymatic reaction network from data. The implications of our findings for other bioinformatics approaches are discussed.

Visualizing plant metabolomic correlation networks using clique-metabolite matrices.

MOTIVATION: Today, metabolite levels in biological samples can be determined using multiparallel, fast, and precise metabolomic approaches. Correlations between the levels of various metabolites can be searched to gain information about metabolic links. Such correlations are the net result of direct enzymatic conversions and of indirect cellular regulation over transcriptional or biochemical processes. In order to visualize metabolic networks derived from correlation lists graphically, each metabolite pair may be represented as vertices connected by an edge. However, graph complexity rapidly increases with the number of edges and vertices. To gain structural information from metabolite correlation networks, improvements in clarity are needed. RESULTS: To achieve this clarity, three algorithms are combined. First, a list of linear metabolite correlations is generated that can be regarded as a set of pairs of edges (or as 2-cliques). Next, a branch-and-bound algorithm was developed to find all maximal cliques by combining submaximal cliques. Due to a clique assignment procedure, the generation of unnecessary submaximal cliques is avoided in order to maintain high efficiency. Differences and similarities to the Bron-Kerbosch algorithm are pointed out. Lastly, metabolite correlation networks are visualized by clique-metabolite matrices that are sorted to minimize the length of lines that connect different cliques and metabolites. Examples of biochemical hypotheses are given that can be built from interpretation of such clique matrices. AVAILABILITY: The algorithms are implemented in Visual Basic and can be downloaded from our web site along with a test data set (http://www.mpimp-golm.mpg.de/fiehn/projekte/data-mining-e.html). CONTACT: kose@mpimp-golm.mpg.de

Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry.

A new liquid chromatography/mass spectrometry (LC/MS) method is described for relative quantification of phosphoproteins to simultaneously compare the phosphorylation status of proteins under two different conditions. Quantification was achieved by beta-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d(5)-thiol selectively at the vinyl moiety of dehydroalanine and dehydroamino-2-butyric acid. The method was evaluated using the model phosphoprotein alpha(S1)-casein, for which three phosphopeptides were found after tryptic digestion. Reproducibility of the relative quantification of seven independent replicates was found to be 11% SD. The dynamic range covered two orders of magnitude, and quantification was linear for mixtures of 0 to 100% alpha(S1)-casein and dephospho-alpha(S1)-casein (R(2) = 0.986). Additionally, the method allowed protein identification and determination of the phosphorylation sites via MS/MS fragmentation.

Biosynthesis of PF1022A and related cyclooctadepsipeptides.

PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2). A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate L-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of D-lactate and D-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the d-hydroxy acid binding site, D-lactate or D-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.

Biosynthesis of taxol: enzymatic acetylation of 10-deacetylbaccatin-III to baccatin-III in crude extracts from roots of Taxus baccata.

The biosynthesis of taxol is a multistep process. One intermediate reaction is the acetylation of 10-deacetylbaccatin-III (10-DAB) to baccatin-III, an assumed precursor of taxol. Here we describe the cell free acetylation of 10-DAB in crude extracts from roots of Taxus baccata saplings using 14C-or 3H-labeled acetyl-coenzyme A as the acetyl donor. The reaction is strictly dependent on the addition of 10-DAB and is specific for the 10-hydroxyl group of the taxane ring. Formation of radiolabeled baccatin-III was confirmed by co-chromatography of the labeled product with authentic baccatin-III in different TLC-systems and HPLC. Furthermore, the acetylation product showed an identical UV spectrum as authentic baccatin-III. Crude extracts from cambium of stems yielded three- to fivefold lower activity. This is in agreement with our finding that the taxol titer in roots was considerably higher than that in cambium.