Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.     

Adaptive,model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells

Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.     

Vascular patterning by Eph receptor tyrosine kinases and ephrins

During vertebrate development, morphologically and functionally very different tissue types and organ systems need to be generated and organised in close coordination with each other. Blood vessels, which become critically required during early embryogenesis and remain indispensable throughout life, need to integrate into a great diversity of tissue types and adapt to both local and systemic requirements of the organism. Far from being randomly placed and uniformly shaped tubes, blood vessels form, with some degree of flexibility, a highly organised and precisely arranged network. Their differentiation, ultrastructure and physiology are well adapted to the requirements and functions of the surrounding tissues. How coordinated development and differentiation are achieved at a molecular level remains to be characterised. This review highlights the large family of Eph receptor tyrosine kinases and their ligands, called ephrins, which, because of their versatile functions in many cell and tissue types and their molecular complexity, might well provide one example of a control system integrating blood vessel and tissue morphogenesis.     

Theoretical and experimental comparisons of gene expression indexes for oligonucleotide arrays

MOTIVATION: Oligonucleotide expression arrays exhibit systematic and reproducible variation produced by the multiple distinct probes used to represent a gene. Recently, a gene expression index has been proposed that explicitly models probe effects, and provides improved fits of hybridization intensity for arrays containing perfect match (PM) and mismatch (MM) probe pairs. RESULTS: Here we use a combination of analytical arguments and empirical data to show directly that the estimates provided by model-based expression indexes are superior to those provided by commercial software. The improvement is greatest for genes in which probe effects vary substantially, and modeling the PM and MM intensities separately is superior to using the PM-MM differences. To empirically compare expression indexes, we designed a mixing experiment involving three groups of human fibroblast cells (serum starved, serum stimulated, and a 50:50 mixture of starved/stimulated), with six replicate HuGeneFL arrays in each group. Careful spiking of control genes provides evidence that 88-98% of the genes on the array are detectably transcribed, and that the model-based estimates can accurately detect the presence versus absence of a gene. The use of extensive replication from single RNA sources enables exploration of the technical variability of the array.     

Confidence measures for protein fold recognition

MOTIVATION: We present an extensive evaluation of different methods and criteria to detect remote homologs of a given protein sequence. We investigate two associated problems: first, to develop a sensitive searching method to identify possible candidates and, second, to assign a confidence to the putative candidates in order to select the best one. For searching methods where the score distributions are known, p-values are used as confidence measure with great success. For the cases where such theoretical backing is absent, we propose empirical approximations to p-values for searching procedures. RESULTS: As a baseline, we review the performances of different methods for detecting remote protein folds (sequence alignment and threading, with and without sequence profiles, global and local). The analysis is performed on a large representative set of protein structures. For fold recognition, we find that methods using sequence profiles generally perform better than methods using plain sequences, and that threading methods perform better than sequence alignment methods. In order to assess the quality of the predictions made, we establish and compare several confidence measures, including raw scores, z-scores, raw score gaps, z-score gaps, and different methods of p-value estimation. We work our way from the theoretically well backed local scores towards more explorative global and threading scores. The methods for assessing the statistical significance of predictions are compared using specificity--sensitivity plots. For local alignment techniques we find that p-value methods work best, albeit computationally cheaper methods such as those based on score gaps achieve similar performance. For global methods where no theory is available methods based on score gaps work best. By using the score gap functions as the measure of confidence we improve the more powerful fold recognition methods for which p-values are unavailable. AVAILABILITY: The benchmark set is available upon request.     

Supporting genotype-phenotype correlation with the rare metabolic diseases database Ramedis

To gain further knowledge about rare genetic diseases, a world wide method for data collection via the Internet has been established. This new approach will improve collecting valuable data from single case reports. Ramedis saves standardised patient data which will be usable for statistics, longitudinal examinations and cooperative studies in future time. Embedded in the scene of the German Human Genome Project, Ramedis directly will enable phenotype-genotype correlations. Beside the better characterisation of clinical heterogeneity of rare metabolic diseases, there may be a great benefit for the treatment of these patients in whom prospective studies are otherwise expensive and difficult to perform. This contribution presents the motivation for this system, introduces features, current state and the future of the project. Additionally, first experiences of using Ramedis by health professionals are explained.     

Metabolomics: quantification of intracellular metabolite dynamics

The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.