Osmotic stress response in Dictyostelium is mediated by cAMP

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium: We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(-) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism.     

The effect of medium-chain triacylglycerols on the blood lipid profile of male endurance runners

Medium-chain triacylglycerol (MCT) oil is currently marketed for athletes as an ergogenic aid for optimal performance. Research assessing the blood lipid response of humans to MCT consumption is very limited and inconclusive. In this randomized cross-over study, male endurance runners (aged 30.5 +/- 5.5 years) were instructed to consume a low-fat diet (approximately 15% of energy) and consume either supplemental MCT oil (30 g twice each day) or long-chain triacylglycerol (LCT) oil (28 g corn oil twice each day) for 14 days. Each dietary trial was separated by at least 3 weeks. At the end of each trial, fasting blood samples were collected and analyzed for serum concentrations of total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), and triacylglycerol (TG). Concentrations of TC (3.83 +/- 0.12 vs. 3.41 +/- 0.15 mmol/L, P = 0.004), LDL-C (1.76 +/- 0.12 vs. 1.51 +/- 0.14 mmol/L, P = 0.033), and TG (1.26 +/- 0.14 vs. 0.98 +/- 0.12 mmol/L, P = 0.006) were higher following the MCT trial than following the LCT trial, respectively. HDL-C concentration did not differ significantly between trials (MCT 1.48 +/- 0.05 mmol/L vs. LCT 1.45 +/- 0.04 mmol/L, P = 0.465). Although blood lipids remained within desirable ranges established by the National Cholesterol Education Program, these results suggest that consumption of MCT oil for 2 weeks negatively alters the blood lipid profile of athletes. Future studies should determine the effects of longer periods of MCT supplementation on serum lipids of exercisers and other groups of individuals. With little data suggesting that MCT are ergogenic, the adverse effects of MCT on blood lipid concentrations may outweigh any proposed benefits for athletes.     

Conversion of solvent evaporation residues from the AB- (acetone - butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters

In a bioconversion study based on utilisation of by-products from the AB- (acetone - butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents. The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp. G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate-(PHA-)accumulation up to 40% per dry weight. A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs. Concentrations of 1-butanol, which is known for its membrane damaging properties in microorganisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth. No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate. Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process. The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.     

Cellular information transfer regarded from a stoichiometry and control analysis perspective

Metabolic control analysis (MCA) allows one to formalize important aspects of information processing in living cells. For example, information processing via multi-level enzyme cascades can be quantified in terms of the response coefficient of a cellular target to a signal. In many situations, control and response coefficients cannot be determined exactly for all enzymes involved, owing to difficulties in 'observing' all enzymes experimentally. Here, we review a number of qualitative approaches that were developed to cope with such situations. The usefulness of the concept of null-space of the stoichiometry matrix for analysing the structure of intracellular signaling networks is discussed. It is shown that signal transduction operates very efficiently when the network structure is such that the null-space matrix can be block-diagonalized (which may or may not imply that the network consists of several disconnected parts) and some enzymes have low elasticities to their substrates.     

Protein expression in yeast; comparison of two expression strategies regarding protein maturation

The driving force for the modification of existing, or the development of new, protein expression systems lies in the identification of a tremendous number of potential novel drug targets through recent genomics approaches. Saccharomyces cerevisiae as a host for recombinant protein expression, offers many advantages, as its biosynthetic pathways resemble higher eukaryotic cells in many aspects. Two yeast vectors were compared to evaluate the versatility of this organism for expression of recombinant proteins. One expression vector enables the secretion of the recombinant protein into the culture medium through fusion with the leader sequence of the mating-type pheromone alpha; the other directs the expression product into the cytoplasm of the yeast cell through fusion with ubiquitin. To facilitate immunological detection and purification, proteins were expressed as fusions to an octapeptide, the so-called Flag-tag, which is recognised by a monoclonal antibody in the presence of Ca2+. We chose 20 functionally different cDNAs to compare the efficiency of both expression systems. All cDNAs could be expressed at the correct size but at varying yields and purity. Both expression systems differed greatly in the degree of glycosylation and other, not further analysed, post-translational modifications. Secretion of all model proteins into the cell culture supernatant could be accomplished if membrane domains or signal sequences were absent, but many proteins were heavily glycosylated as demonstrated by lectin mapping or enzymatical deglycosylation. Some proteins, however, were expressed as homogenous products, and could be easily purified for further functional studies. Further investigations on the expression biology of yeast are required, in order to optimise the conditions of fermentation which may finally lead to more homogeneous expression products.     

Computer-Aided Resolution of an Experimental Paradox in Bacterial Chemotaxis

Escherichia coli responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190–5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703–8707, 1989). For example, “building up” the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas “breaking down” the pathway by deleting chemotaxis genes except cheA and cheY resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the che genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks.