Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110

<p>Abstract</p> <p>Background</p> <p>Norvaline is an unusual non-proteinogenic branched-chain amino acid which has been of interest especially during the early enzymological studies on regulatory mutants of the branched-chain amino acid pathway in <it>Serratia marcescens</it>. Only recently norvaline and other modified amino acids of the branched-chain amino acid synthesis pathway got attention again when they were found to be incorporated in minor amounts in heterologous proteins with a high leucine or methionine content. Earlier experiments have convincingly shown that norvaline and norleucine are formed from pyruvate being an alternative substrate of α-isopropylmalate synthase, however so far norvaline accumulation was not shown to occur in non-recombinant strains of <it>E. coli</it>.</p> <p>Results</p> <p>Here we show that oxygen limitation causes norvaline accumulation in <it>E. coli </it>K-12 W3110 during grow in glucose-based mineral salt medium. Norvaline accumulates immediately after a shift to oxygen limitation at high glucose concentration. On the contrary free norvaline is not accumulated in <it>E. coli </it>W3110 in aerobic cultures. The analysis of medium components, supported by transcriptomic studies proposes a purely metabolic overflow mechanism from pyruvate into the branched chain amino acid synthesis pathway, which is further supported by the significant accumulation of pyruvate after the oxygen downshift. The results indicate overflow metabolism from pyruvate as necessary and sufficient, but deregulation of the branched chain amino acid pathway may be an additional modulating parameter.</p> <p>Conclusion</p> <p>Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over α-ketobutyrate and α-ketovalerate.</p> <p>Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiology of <it>E. coli </it>under oxygen limitation in general.</p

Transient increase of ATP as a response to temperature up-shift in Escherichia coli

BACKGROUND: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift. RESULTS: The current study shows that a temperature up-shift is followed by a very fast significant transient increase of the cellular ATP concentration within the first minutes. This increase is connected to a longer lasting elevation of the cellular respiration and glucose uptake. Also the mRNA level of typical heat shock genes increases within only one minute after the heat-shock. CONCLUSION: The presented data prove the very fast response of E. coli to a heat-shock and that the initial response includes the increase of the ATP pool which is important to fulfil the need of the cell for new syntheses, as well as for the function of chaperones and proteases

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma

Identification and functional analysis of novel components of the TRAIL apoptosis signalling pathway

Apoptosis, a controlled form of cell death, plays a central role in the development and homeostasis of multicellular organisms. Apoptosis induction can occur via intracellular mechanisms, for example after intensive DNA damage, or via triggering of death receptors. Death receptors belong to the tumour necrosis factor (TNF) receptor superfamily. Amongst the death receptors, the TNF-related apoptosisinducing ligand (TRAIL) and its five receptors stand out due to its complexity and tumour-specific killing activity. TRAIL can bind two apoptosis-inducing receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), two additional cell-bound receptors incapable of transmitting an apoptotic signal, TRAIL-R3 (LIT, DcR1) and TRAIL-R4 (TRUNDD, DcR2), sometimes also called decoy receptors, and lastly, a soluble receptor called osteoprotegerin (OPG). TRAIL has been shown to induce apoptosis in cancer cells in vivo without causing toxicity. On the basis of these findings, specific activation of TRAIL-R1 and TRAILR2 is currently evaluated for tumour therapy in early clinical trials. Yet, primary tumours are mostly resistant to TRAIL-induced apoptosis. Interestingly however, chemo- and radiotherapy can sensitise primary tumour cells to TRAIL-induced apoptosis whereas normal cells remain resistant also to such combinations. To understand the molecular mechanisms of tumour-cell-specific sensitisation to TRAIL, genome-wide RNA interference (RNAi) was used in an unbiased approach with the aim to identify novel factors involved in TRAIL-induced apoptosis and its inhibition. By screening the TRAIL-sensitive human cervix carcinoma cell line HeLa novel modulators of TRAIL signalling were identified. The studies presented in this thesis focus on the identification and validation of factors that are required for TRAIL apoptosis signalling, the molecular characterisation and the determination of their biochemical mechanism of action

Response of mRNAs in a stirred-flask cultivation of W3110 with a temperature shock from 30°C to 42°C

<p><b>Copyright information:</b></p><p>Taken from "Transient increase of ATP as a response to temperature up-shift in "</p><p>Microbial Cell Factories 2005;4():9-9.</p><p>Published online 1 Apr 2005</p><p>PMCID:PMC1087501.</p><p>Copyright © 2005 Soini et al; licensee BioMed Central Ltd.</p> For experimental details see Materials and Methods

Response of the adenosine nucleotide pool to a temperature up-shift from 30 to 42°C in W3110

<p><b>Copyright information:</b></p><p>Taken from "Transient increase of ATP as a response to temperature up-shift in "</p><p>Microbial Cell Factories 2005;4():9-9.</p><p>Published online 1 Apr 2005</p><p>PMCID:PMC1087501.</p><p>Copyright © 2005 Soini et al; licensee BioMed Central Ltd.</p> (A) ATP and cultivation temperature, (B) ADP, (C) AMP. The data were obtained during the batch cultivation shown in Figure 1

Batch cultivation of W3110 with a temperature up-shift performed at time 0 by switching the temperature set-point from 30 to 42°C

<p><b>Copyright information:</b></p><p>Taken from "Transient increase of ATP as a response to temperature up-shift in "</p><p>Microbial Cell Factories 2005;4():9-9.</p><p>Published online 1 Apr 2005</p><p>PMCID:PMC1087501.</p><p>Copyright © 2005 Soini et al; licensee BioMed Central Ltd.</p> (A) OD(○), temperature (—); (B) glucose (□), acetate (△); (C) q(○), q(▽), RQ (□), (D) pH (△), relative units of ammonia added (+). The grey area indicates the time period of about 5 min during which the temperature increased

Dynamics of the Energy Charge and sum of adenosine phosphates (AXP) during a cultivation of W3110 with temperature up-shift from 30 to 42°C

<p><b>Copyright information:</b></p><p>Taken from "Transient increase of ATP as a response to temperature up-shift in "</p><p>Microbial Cell Factories 2005;4():9-9.</p><p>Published online 1 Apr 2005</p><p>PMCID:PMC1087501.</p><p>Copyright © 2005 Soini et al; licensee BioMed Central Ltd.</p> Data were calculated from primary concentrations of the adenosine nucleotides shown in Figure 2