Experimental optimization of the optical and electrical properties of a half-wavelength-thick organic hetero-structure in a Micro-cavity.

In the context of progress towards the organic laser diode, we experimentally investigate the optical and electrical optimization of an OLED in a vertical λ/2 microcavity. The microcavity consists of a quarter-wavelength TiO2/SiO2 multilayer mirror, a half-wavelength-thick OLED and a semitransparent aluminum cathode. The Alq3/DCM2 guest-host system is used as the emitting layer. This study focuses on the design and the fabrication of a half-wavelength thick organic hetero-structure exhibiting a high current density despite both the thickness increase and the cathode thickness reduction. The emission wavelength, the line-width narrowing and the current-density are studied as a function of two key parameters: the hetero-structure optical thickness and the aluminum cathode thickness. The experimental results show that a 125 nm thick cavity OLED ended by a 20 nm thick aluminum cathode exhibits a resonance at 606 nm with a full width at half maximum of 11 nm, and with current-densities exceeding 0.5 A/cm². We show that even without a top high-quality-mirror the incomplete microcavity λ/2 OLED hetero-structure exhibits a clear modification of the spontaneous emission at normal incidence.

Feedback phase sensitivity of a semiconductor laser subject to filtered optical feedback: experiment and theory.

We investigate experimentally and theoretically the dynamics of a semiconductor laser subject to filtered optical feedback. Depending on the feedback strength we find dynamical regimes with different dependence on the feedback phase. In particular, the influence of the feedback phase on cw emission and on frequency oscillations is characterized experimentally. We also measure the dependence of the filter mirror distance on the frequency oscillations. In general, good agreement between experiment and theory is found.

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae.

Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as "overflow metabolism" or "the Crabtree effect." The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.

Frequency versus relaxation oscillations in a semiconductor laser with coherent filtered optical feedback.

We investigate the dynamics of a semiconductor laser subject to coherent delayed filtered optical feedback. A systematic bifurcation analysis reveals that this system supports two fundamentally different types of oscillations, namely relaxation oscillations and external roundtrip oscillations. Both occur stably in large domains under variation of the feedback conditions, where the feedback phase is identified as a key quantity for controlling this dynamical complexity. We identify two separate parameter regions of stable roundtrip oscillations, which occur throughout in the form of pure frequency oscillations.

Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio.

Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.

Increased recombinant protein production in Escherichia coli strains with overexpressed water-forming NADH oxidase and a deleted ArcA regulatory protein.

Glycolytic flux is increased and acetate production is reduced in Escherichia coli by the expression of heterologous NADH oxidase (NOX) from Streptococcus pneumoniae coupled with the deletion of the arcA gene, which encodes the ArcA regulatory protein. In this study, we examined the overproduction of a model recombinant protein in strains of E. coli expressing NOX with or without an arcA mutation. The presence of NOX or the absence of ArcA reduced acetate by about 50% and increased beta-galactosidase production by 10-20%. The presence of NOX in the arcA strain eliminated acetate production entirely in batch fermentations and resulted in a 120% increase in beta-galactosidase production.

Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase.

We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth.

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions.

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h.

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli.

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.

Oligodendrocytes and their precursors require phosphatidylinositol 3-kinase signaling for survival.

Signal transduction in response to several growth factors that regulate oligodendrocyte development and survival involves the activation of phosphatidylinositol 3-kinase, which we detect in oligodendrocytes and their precursors. To investigate the role of this enzyme activity, we analyzed cell survival in cultures of oligodendrocytes treated with wortmannin or LY294002, two potent inhibitors of phosphatidylinositol 3-kinase. Cell survival was inhibited by 60-70% in these cultures within 24 hours, as quantitated by a tetrazolium staining assay for viable cells and by measurement of DNA content. Similar results were obtained with oligodendrocyte precursor cells. Nuclei of the dying cells contained fragmented DNA, as revealed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays, indicating that the cells were dying by apoptosis. Moreover, a significant increase in the number of cells with fragmented nuclear DNA was detected as early as 4 hours, well before any significant differences could be detected in glucose transport or cell viability. Exogenous addition of insulin-like growth factor-I, neurotrophin-3, platelet-derived growth factor, basic fibroblast growth factor, ciliary neurotrophic factor, N-acetyl cysteine, vitamin C, vitamin E, progesterone or serum did not prevent cell death in the presence of wortmannin or LY294002. These findings indicate that survival of oligodendrocytes and their precursors depends on a phosphatidylinositol 3-kinase mediated signaling pathway. Inhibition of this critical enzyme activity induces apoptotic cell death, even in the presence of exogenous growth factors or serum.

Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells.

We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane 'blebs', detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The 'blebs' are distinguishable from 'ruffles' or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5'[gamma-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(gamma) and p85/PI 3-K, regulated by G beta gamma subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stiumlated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 approximately 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 microM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.

Topology of Wolfgram proteins and 2', 3'-cyclic nucleotide 3'-phosphodiesterase in CNS myelin: studies with proteases.

The topological disposition of Wolfgram proteins (WP) and their relationship with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W > 14KDa were detected on the gels. Therefore protein fragments of WP with M.W < 14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.