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C Wittmann

Publications and source records attributed to C Wittmann.

At least 19 recordsLinked to original sources
PubMed2002

Metabolic flux analysis using mass spectrometry.

Detailed knowledge on carbon flux distributions is crucial for the understanding and targeted optimization of cellular systems. Analytical methods to identify the topology of metabolic networks and to quantify fluxes through its different pathways are therefore in the core of metabolic engineering. An elegant approach for metabolic flux analysis is provided by tracer experiments. In such studies tracer substrates with stable isotopes such as 13C are applied and the labeling pattern of metabolites is subsequently measured. Detailed flux distributions can be obtained by a combination of tracer experiments and stoichiometric balancing. In recent years, mass spectrometry (MS) has emerged as an interesting method for labeling measurements in metabolic flux analysis and provided valuable insights into the cellular metabolism. The present review provides an overview on current experimental and modeling tools for metabolic flux analysis by MS. The application of MS for flux analysis is illustrated by examples from the literature for various biological systems, including bacteria, fungi, tissue cultures and in vivo studies in humans.

Carbon Isotopes↗
PubMed2002

Ecology and ecophysiology of tree stems: corticular and wood photosynthesis.

Below the outer peridermal or rhytidomal layers, most stems of woody plants possess greenish tissues. These chlorophyll-containing tissues (the chlorenchymes) within the stems are able to use the stem internal CO2 and the light penetrating the rhytidome to photoassimilate and produce sugars and starch. Although net photosynthetic uptake of CO2 is rarely found, stem internal re-fixation of CO2 in young twigs and branches may compensate for 60-90% of the potential respiratory carbon loss. Isolated chlorenchymal tissues reveal rather high rates of net photosynthesis (being up to 75% of the respective rates for leaf photosynthesis). Corticular photosynthesis is thus thought to be an effective mechanism for recapturing respiratory carbon dioxide before it diffuses out of the stem. Furthermore, chloroplasts of the proper wood or pith fraction also take part in stem internal photosynthesis. Although there has been no strong experimental evidence until now, we suggest that the oxygen evolved during wood or pith photosynthesis may play a decisive role in avoiding/reducing stem internal anaerobiosis.

Carbon Dioxide↗
PubMed2002

Influence of glucose, fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by Corynebacterium glutamicum.

Batch cultivations of l-lysine-producing Corynebacterium glutamicum ATCC 21253 were carried out on the different carbon sources, glucose, sucrose and fructose. The time profiles of substrate and product concentrations were evaluated to compare kinetics and stoichiometry of lysine production. The lysine yield (mol C/mol C) on glucose was 8% higher than on sucrose and 30% higher than on fructose. The highest final biomass concentration of 5.0 g/l was obtained on glucose, whereas fructose and sucrose yielded 20% less biomass. Compared to glucose, fructose resulted in significantly higher respiration rates, a higher substrate uptake rate but a lower lysine production rate during the cultivation process. This was probably due to a higher tricarboxylic cycle activity combined with a lower activity of the pentose phosphate pathway. On sucrose, specific rates and yields differed significantly from those on fructose and glucose. Transport and metabolism of sucrose, therefore, are not a simple superposition of its building blocks, glucose and fructose.

Biomass↗
PubMed2001

MALDI-TOF MS for quantification of substrates and products in cultivations of Corynebacterium glutamicum.

The application of MALDI-TOF MS for the quantification of lysine, alanine, and glucose is described. The method is based on using stable isotopes as internal standards and allows fast, sensitive, and reproducible quantification of these compounds. It is demonstrated for aqueous standard solutions with concentrations of the analytes between 10 microM and 100 mM. The mean standard deviations from five replicates each were 4.3% (lysine), 3.7% (alanine), and 3.2% (glucose). In addition, sucrose could be measured by MALDI-TOF MS, but was not quantified due to lack of an internal standard. The method developed can be applied to quantify metabolites in cultivations of C. glutamicum ATCC 21253, without processing of the sample except a 1:5 dilution. Excellent agreement of the data with conventional techniques like HPLC or enzyme assay was found.

Cell Culture Techniques↗
PubMed2001

Modeling and experimental design for metabolic flux analysis of lysine-producing Corynebacteria by mass spectrometry.

Experimental design of (13)C-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios. Ratio measurement of mass isotopomer pools of Corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (Phi(PPP)), (ii) between the split pathways in the lysine biosynthesis (Phi(DH)), (iii) at the pyruvate node (Phi(PC)), and reversibilities of (iv) glucose 6-phosphate isomerase (zeta(PGI)), (v) at the pyruvate node (zeta(PC/PEPCK)), and (vi) of transaldolase and transketolases in the PPP. Weighted sensitivities for flux parameters were derived from partial derivatives to quantitatively evaluate experimental approaches and predict precision for estimated flux parameters. Deviation of intensity ratios from ideal values of 1 was used as weighting function. Weighted flux sensitivities can be used to identify optimal type and degree of tracer labeling or potential intensity ratios to be measured. Experimental design for lysine-producing strain C. glutamicum MH 20-22B (Marx et al., Biotechnol. Bioeng. 49, 111-129, 1996) and various potential mutants with different alterations in the flux pattern showed that specific tracer labelings are optimal to quantify a certain flux parameter uninfluenced by the overall flux situation. Identified substrates of choice are [1-(13)C]glucose for the estimation of Phi(PPP) and zeta(PGI) and a 1 : 1 mixture of [U-(12)C/U-(13)C]glucose for the determination of zeta(PC/PEPCK). Phi(PC) can be quantified by feeding [4-(13)C]glucose or [U-(12)C/U-(13)C]glucose (1 : 1), whereas Phi(DH) is accessible via [4-(13)C]glucose. The sensitivity for the quantification of a certain flux parameter can be influenced by superposition through other flux parameters in the network, but substrate and measured mass isotopomers of choice remain the same. In special cases, reduced labeling degree of the tracer substrate can increase the precision of flux analysis. Enhanced precision and flux information can be achieved via multiply labeled substrates. The presented approach can be applied for effective experimental design of (13)C tracer studies for metabolic flux analysis. Intensity ratios of other products such as glutamate, valine, phenylalanine, and riboflavin also sensitively reflect flux parameters, which underlines the great potential of mass spectrometry for flux analysis.

Computer Simulation↗
PubMed2001

Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae.

The dynamics of intracellular amino acid pools were determined in batch cultures of Saccharomyces cerevisiae. Immediate termination of metabolic activity was found to be necessary for accurate quantification of in vivo concentrations of intracellular amino acids, due to significant changes in most intracellular amino acid pools observed during extraction without an instantaneous stop of the metabolism. The method applied to batch-cultures of S. cerevisiae on glucose revealed complex dynamics in intracellular amino acid pools. The most drastic changes were observed during the diauxic shift and at the entry into the stationary phase. Even during phases of exponential growth on glucose and ethanol, cells showed significant variations in intracellular amino acid concentrations. The method presented can be used to investigate the physiology of yeast cultures, including industrially relevant batch and fed-batch processes.

Amino Acids↗
PubMed2001

Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum: a novel approach for metabolic flux analysis.

In the present work, a novel comprehensive approach of (13)C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 microL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-(13)C glucose or with mixtures of (13)C6/(12)C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.

Citric Acid Cycle↗
PubMed2000

Evaluation of ecological disturbance and intrinsic bioremediation potential of pulp mill-contaminated lake sediment using key enzymes as probes.

A rapid protocol was developed to measure 10 different enzymic activities from a large number of 1-cm-sliced freshly collected lake sediments. Layers heavily polluted by organic halogens (4900 mg Cl kg(-1)) revealed severe depression of phosphatase, sulfatase, leucine-aminopeptidase, chitinase, acetate esterase and butyrate esterase activities as compared to layers above and below the most polluted zone. alpha-Glucosidase, beta-glucosidase, beta-xylosidase and palmitate esterase were less affected. Methane oxidation potential was dramatically depressed in the polluted strata whereas tetrachloromethane dehalogenating activity was observed in the polluted sediment only. The sediment layers formed after the chlorine discharges into the lake had diminished to 1/10, and showed restoration of the activities close to those observed in non-recipient sediment, in spite of the persisting presence of >1000 mg of organic chlorine (kg dry wt)(-1). We conclude that certain enzymic activities involved in breakdown or oxidation of organic matter in the sediments are useful probes for assessing the degree of ecological damage and its potential for restoration in recipient lakes of industrial discharges.

Journal Article↗
PubMed2000

Radon and thoron in cave dwellings (Yan'an, China).

222Rn and 220Rn concentrations were measured in cave dwellings and brick houses in the region of Yan'an (China) during summer 1997. The underground dwellings are built into Quaternary loess, and all investigated houses are founded on it. The median values of indoor 222Rn and 220Rn concentrations are 42 (n = 18) and 77 Bq m(-3) (n = 15) for brick houses and 92 (n = 23) and 215 (n = 17) Bq m(-3) for cave dwellings. To classify the dwellings in respect to their "cave-character," the fraction of walls having a direct contact to the loses is calculated for each dwelling. While the 222Rn concentrations are increasing with higher fractions, the 220Rn concentrations are not correlated with this fraction. On the other hand, due to the short half-life of 220Rn the distance from the measuring point to the walls is negatively correlated with the 220Rn concentration, while there is no correlation with the 222Rn concentration. Therefore, concentric isolines of 220Rn concentrations showing a strong gradient were detected in cave dwellings. An influence of the ventilation rate is distinct for 222Rn but weak for 220Rn. The effective dose rates for 222Rn and 220Rn and their progenies are calculated for brick houses (2.7 mSv y(-1)), cave dwellings (7.1 mSv y(-1)), and for traditional cave dwellings with a bed foundation built with loess (16.7 mSv y(-1)). These calculations are based on summer measurements only. It is expected that the true effective dose rates will be significantly higher.

China↗
PubMed1999

Mass spectrometry for metabolic flux analysis.

Mass spectrometry in combination with tracer experiments based on 13C substrates can serve as a powerful tool for the modeling and analysis of intracellular fluxes and the investigation of biochemical networks. The theoretical background for the application of mass spectrometry to metabolic flux analysis is discussed. Mass spectrometry methods are especially useful to determine mass distribution of metabolites. Additional information gained from fragmentation of metabolites, e.g., by electron impact ionization, allows further localization of labeling positions, up to complete resolution of isotopomer pools. To effectively handle mass distributions in simulation experiments, a matrix based general methodology is formulated. The natural isotope distribution of carbon, oxygen, hydrogen and nitrogen in the target metabolites is considered by introduction of correction matrices. It is shown by simulation results for the central carbon metabolism that neglecting natural isotope distributions causes significant errors in intracellular flux distributions. By varying relative fluxes into pentosephosphate pathway and pyruvate carboxylation reaction, marked changes in the mass distributions of metabolites result, which are illustrated for pyruvate, oxaloacetate, and alpha-ketoglutarate. In addition mass distributions of metabolites are significantly influenced over a broad range by the degree of reversibility of transaldolase and transketolase reactions in the pentosephosphate pathway. The mass distribution of metabolites is very sensitive towards intracellular flux patterns and can be measured with high accuracy by routine mass spectrometry methods. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗
PubMed1999

Analysis of terbuthylazine in soil samples by two test strip immunoassay formats using reflectance and luminescence detection.

Two different immunoassay (IA) formats for terbuthylazine analysis were developed and optimized using the same monoclonal antibodies. The measuring range for the dipstick IA (using an alkaline phosphatase tracer, a 5-bromo-4-chloroindolylphosphate/nitro blue tetrazolium substrate and a portable reflectometer) was from 3 to 300 microg/kg. The teststrip IA (investigating a horseradish peroxidase tracer, a luminol substrate, and a portable luminometer prototype developed by Immunotek, Moscow, Russia) had a measuring range from 0.05 to 10 microg/kg. From 24 soil samples collected in the Veneto area, Italy, 18 samples contained different amounts of terbuthylazine (but <1 microg/kg atrazine) as was analyzed by gas chromatography with mass selective detection. Eight soil samples (six positive, two negative controls) were analyzed according to the two IA formats. Whereas the dipstick with reflectance detection yielded satisfying results, the test strip IA using luminescence detection has failed so far for soil samples.

Chromatography, Gas↗
PubMed1999

Anterior organization of the Caenorhabditis elegans embryo by the labial-like Hox gene ceh-13.

The Caenorhabditis elegans lin-39, mab-5 and egl-5 Hox genes specify cell fates along the anterior-posterior body axis of the nematode during postembryonic development, but little is known about Hox gene functions during embryogenesis. Here, we show that the C. elegans labial-like gene ceh-13 is expressed in cells of many different tissues and lineages and that the rostral boundary of its expression domain is anterior to those of the other Hox genes. By transposon-mediated mutagenesis, we isolated a zygotic recessive ceh-13 loss-of-function allele, sw1, that exhibits an embryonic sublethal phenotype. Lineage analyses and immunostainings revealed defects in the organization of the anterior lateral epidermis and anterior body wall muscle cells. The epidermal and mesodermal identity of these cells, however, is correctly specified. ceh-13(sw1) mutant embryos also show fusion and adhesion defects in ectodermal cells. This suggests that ceh-13 plays a role in the anterior organization of the C. elegans embryo and is involved in the regulation of cell affinities.

Amino Acid Sequence↗
PubMed1997

Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos.

In a 4-cell stage C. elegans embryo, signaling by the P2 blastomere induces anterior-posterior polarity in the adjacent EMS blastomere, leading to endoderm formation. We have taken genetic and reverse genetic approaches toward understanding the molecular basis for this induction. These studies have identified a set of genes with sequence similarity to genes that have been shown to be, or are implicated in, Wnt/Wingless signaling pathways in other systems. The C. elegans genes described here are related to wnt/wingless, porcupine, frizzled, beta-catenin/armadillo, and the human adenomatous polyposis coli gene, APC. We present evidence that there may be partially redundant inputs into endoderm specification and that a subset of these genes appear also to function in determining cytoskeletal polarity in certain early blastomeres.

Adenomatous Polyposis Coli Protein↗
PubMed1997

The expression of the C. elegans labial-like Hox gene ceh-13 during early embryogenesis relies on cell fate and on anteroposterior cell polarity.

Clusters of homeobox-containing HOM-C/hox genes determine the morphology of animal body plans and body parts and are thought to mediate positional information. Here, we describe the onset of embryonic expression of ceh-13, the Caenorhabditis elegans orthologue of the Drosophila labial gene, which is the earliest gene of the C. elegans Hox gene cluster to be activated in C. elegans development. At the beginning of gastrulation, ceh-13 is asymmetrically expressed in posterior daughters of anteroposterior divisions, first in the posterior daughter of the intestinal precursor cell E and then in all posterior daughters of the AB descendants ABxxx. In this paper, we present evidence that supports position-independent activation of ceh-13 during early C. elegans embryogenesis, which integrates cell fate determinants and cell polarity cues. Our findings imply that mechanisms other than cell-extrinsic anteroposterior positional signals play an important role in the activation and regulation of the C. elegans Hox gene ceh-13.

Animals↗
PubMed1996

cec-1, a soma-specific chromobox-containing gene in C. elegans.

The chromo domain is a phylogenetically conserved sequence motif which was identified as a region of homology between the repressor protein Pc and the heterochromatin constitutive protein HP1 of Drosophila. The specific function of the chromo domain is not yet understood, but it seems to be required for protein-protein interactions in chromatin-associated complexes. Here, we present a new chromobox-containing gene from Caenorhabditis elegans (cec-1). It encodes a nuclear protein that is present in all somatic cells from the 50- to 80-cell stage on throughout development and in adult animals. No cec-1 protein was detected in the cells of early embryos, in germ cells, and in their precursor cells Z2 and Z3. cec-1 mRNA, however, is already present in all the blastomeres of early embryos. Immunolocalization experiments revealed a homogeneous distribution of CEC-1 within interphase nuclei, while during mitosis CEC-1 seems to dissociate from the condensing chromosomes. The expression pattern of the cec-1 gene suggests that it may represent a new regulatory gene in C. elegans.

Amino Acid Sequence↗
PubMed1996

Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site.

On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.

Atrazine↗
PubMed1995

Growth inhibition by ammonia and use of a pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum.

The inhibitory effect of ammonia on the growth of the polychlorinated xenobiotic-degrading bacterium Mycobacterium chlorophenolicum was examined. The strain is inhibited by both the ionized and nonionized forms of ammonia, At pH 6.9, 50% reduction of the growth rate was observed at 6.8 g/l total ammonium. For 23 experiments performed in shake-flask culture at different pH values, and ammonium concentrations a growth model based on the extended Monod kinetic fits the data with a deviation of 5.3%. To overcome growth inhibition in bioreactors, a pH- controlled feeding strategy was developed for effective cultivation of M. chlorophenolicum at an ammonium level below 0.3 g/l. The ammonium addition was controlled online by the stoichiometric interdependence of ammonium consumption and pH decline. With this online control strategy, a biomass concentration as high as 26.2 g/l can be achieved within less than a week of cultivation. The yield is also increased from 0.32 g to 0.43 g biomass (per gram glucose). The strategy developed provides an effective method for the production of biomass o M. chlorophenolicum serving as the inoculum in remediation technologies.

Automation↗