Integration of metabolite with transcript and enzyme activity profiling during diurnal cycles in Arabidopsis rosettes

ABSTRACT: BACKGROUND: Genome-wide transcript profiling and analyses of enzyme activities from central carbon and nitrogen metabolism has shown that transcript levels undergo marked and rapid changes during diurnal cycles and after transfer to darkness, whereas changes of enzyme activities are smaller and delayed. In the starchless pgm mutant, where sugars are depleted every night, there are accentuated diurnal changes of transcript levels. Enzyme activities do not show larger diurnal changes; instead they shift towards the levels found in wild-type after several days of darkness. These results indicate that enzyme activities change slowly, integrating the changes of transcript levels over several diurnal cycles. RESULTS: To generalize this conclusion, 137 metabolites were profiled using GC-MS and LC-MS. Amplitudes of the diurnal changes of metabolites in pgm were (with the exception of sugars) similar or smaller than in wild-type. The average levels shifted towards those found after several days of darkness in wild-type. Examples include increased levels of many amino acids due to protein degradation, decreased levels of many fatty acids, increased tocopherol and decreased myo-inositol. Many metabolite-transcript correlations were found and the proportion of transcripts correlated with sugars increased dramatically in the starchless pgm mutant. CONCLUSION: Rapid diurnal changes of transcripts are integrated over time to generate quasi-stable changes across large sectors of metabolism. The slow response of enzyme activities and metabolites implies that correlations between metabolites and transcripts are due to regulation of gene expression by metabolites, rather than metabolites being changed as a consequence of a change in gene expression

Determination of the prebiotic activity of wheat arabinogalactan peptide (AGP) using batch culture fermentation

Purpose To test the prebiotic activity of wheat arabinogalactan-peptide (AGP), which is a soluble dietary fibre composed of arabinogalactan polysaccharide linked to a 15-residue peptide, which accounts for up to 0.4% of the dry weight of wheat flour. Methods The prebiotic activity of AGP prepared from white wheat flour was tested using in-vitro fermentation by colonic bacteria in automated pH controlled anaerobic stirred batch cultures and compared to fructooligosaccharide (FOS) and wheat flour arabinoxylan (AX). Bacterial populations were measured using fluorescence in-situ hybridisation (flow-FISH) and short-chain fatty acid (SCFA) concentrations were measured using HPLC. Results Fermentation of AGP resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate after 24 h of fermentation. Conclusions These results were comparable to those obtained with AX and confirm the prebiotic potential of AGP. Furthermore, fermentation of a mixture of AGP and AX was faster compared to the single substrates and more similar to FOS, indicating that combinations of fermentable carbohydrates with different structures are potentially more effective as prebiotics than single substrates

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain Gö1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain Gö1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide

Isolation of a cytochrome-deficient mutant strain of Sporomusa sphaeroides not capable of oxidizing methyl groups.

The homoacetogenic anaerobic bacterium Sporomusa sphaeroides was mutagenized with UV light. Taking advantage of the ampicillin enrichment technique and a newly developed test for the detection of heme in bacterial colonies, the cytochrome-deficient mutant strain S. sphaeroides BK824 was isolated. In contrast to the wild type, this mutant strain failed to grow on betaine, betaine plus methanol, H2 plus CO2, and methanol plus CO2. Growth on betaine plus formate, betaine plus H2, betaine plus pyruvate, methanol plus H2 and CO2, and acetoin was not impaired. All enzymes of the Wood pathway as well as hydrogenase and carbon monoxide dehydrogenase were detectable at comparable activities in both the wild type and the cytochrome-deficient mutant. Labeling experiments with [14C]methanol demonstrated the inability of S. sphaeroides BK824 to oxidize methyl groups. The role of cytochromes in electron transport steps associated with the Wood pathway enzymes and their possible role in energy conservation during autotrophic growth in acetogens are discussed

Sarcosine in prostate cancer tissue is not a differential metabolite for prostate cancer aggressiveness and biochemical progression

PURPOSE: Sarcosine in prostate cancer tissue samples was recently reported to be increased during prostate cancer progression to metastasis and suggested to be a key metabolite of cancer cell invasion and aggressiveness. We reevaluated sarcosine in prostate cancer tissue samples as a potential indicator of tumor aggressiveness, and as a predictor of recurrence-free survival. MATERIALS AND METHODS: Sarcosine in matched samples of malignant and nonmalignant tissue from 92 patients with prostate cancer after radical prostatectomy was measured in the framework of a global metabolite profiling study of prostate cancer by gas chromatography/mass spectrometry. We related results to age, prostate volume, tumor stage, Gleason score, preoperative prostate specific antigen and biochemical recurrence, defined as a persistent prostate specific antigen increase of greater than 0.2 ng/ml. Nonparametric statistical tests, ROC curves and Kaplan-Meier analyses were done. RESULTS: Median sarcosine content in tissue was about 7% higher in matched malignant vs nonmalignant samples, which was significantly. Sarcosine values were not associated with tumor stage (pT2 vs pT3), tumor grade (Gleason score less than 7 vs 7 or greater) or biochemical recurrence. The lack of metastatic tissue samples was a study limitation. CONCLUSIONS: Sarcosine in prostate cancer tissue samples cannot be considered a suitable predictor of tumor aggressiveness or biochemical recurrence. Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved

Multilevel genomic analysis of the response of transcripts, enzyme activities and metabolites in Arabidopsis rosettes to a progressive decrease of temperature in the non-freezing range

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Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings

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