The arabinose-inducible promoter P BAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-␤-D-thiogalactopyranoside-inducible P tac and P taclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P BAD ) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.In 1957, Novick and Weiner (14) studied expression of the lac operon in the presence of inducer concentrations less than that needed for maximal induction (subsaturating concentrations). This early study demonstrated that a fraction of cells in the population was fully induced while the remainder was uninduced and that the number of fully induced cells varied directly with the concentration of inducer. They referred to this mechanism as "all-or-none" or autocatalytic gene expression (14). Autocatalytic gene expression systems contain the genes encoding the transporter under the control of the transported molecule (the inducer). More recently, autocatalytic behavior was also reported for the ara operon (18).Although the all-or-none phenomenon associated with autocatalytic expression systems was demonstrated more than 40 years ago, many of the expression systems currently available continue to be based on similar frameworks and used without regard for this phenomenon. For systems in which population heterogeneity is not important and high-level gene expression is desired, autocatalytic systems remain an ideal choice; expression can be induced to a maximal level in all cells of the populat...

Invasiveness is a hallmark of aggressive cancer like malignant melanoma, and factors involved in acquisition or maintenance of an invasive phenotype are attractive targets for therapy. We investigated melanoma phenotype modulation induced by the metastasis-promoting microenvironmental protein S100A4, focusing on the relationship between enhanced cellular motility, dedifferentiation and metabolic changes. In poorly motile, well-differentiated Melmet 5 cells, S100A4 stimulated migration, invasion and simultaneously down-regulated differentiation genes and modulated expression of metabolism genes. Metabolic studies confirmed suppressed mitochondrial respiration and activated glycolytic flux in the S100A4 stimulated cells, indicating a metabolic switch toward aerobic glycolysis, known as the Warburg effect. Reversal of the glycolytic switch by dichloracetate induced apoptosis and reduced cell growth, particularly in the S100A4 stimulated cells. This implies that cells with stimulated invasiveness get survival benefit from the glycolytic switch and, therefore, become more vulnerable to glycolysis inhibition. In conclusion, our data indicate that transition to the invasive phenotype in melanoma involves dedifferentiation and metabolic reprogramming from mitochondrial oxidation to glycolysis, which facilitates survival of the invasive cancer cells. Therapeutic strategies targeting the metabolic reprogramming may therefore be effective against the invasive phenotype.

This study demonstrates for the first time the potential of HR-MAS (1)H NMR spectroscopy as an analytical tool for use on intact lenses. Near-threshold UVR-B doses led to a generally significant decrease in water-soluble metabolites 1 week after exposure. The lack of dose-dependent changes in the metabolites indicates that repair processes during the first week after UVB irradiation overcome the immediate metabolic disturbances.

Abbreviations used: b-HOB, b-hydroxybutyrate; LC-MS, liquid chromatography mass spectrometry; MAS, malate-aspartate shuttle; MCL, molecular carbon labeling; MCT, monocarboxylate transporter. AbstractKetone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-13 C]b-hydroxybutyrate to that of [1,6-13 C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate-glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of 13 C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-13 C]b-hydroxybutyrate as opposed to [1,6-13 C]glucose. Our results suggest that the change in aspartate-glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate-aspartate shuttle activity in neurons using b-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only bhydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing bhydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate-aspartate shuttle.