Fibroblast growth factor signalling and regional specification of the pharyngeal ectoderm

Branchial arch development involves dynamic interactions between neural crest cells as well as ectodermal, endodermal and mesodermal cell populations. Despite their importance and evolutionary conservation, the intercellular interactions guiding the early development of the branchial arches are still poorly understood. We have here studied fibroblast growth factor (FGF) signalling in early pharyngeal development. In mice homozygous for a hypomorphic allele of Fgfr1, neural crest cells migrating from the hindbrain mostly fail to enter the second branchial arch. This defect is non-cell-autonomous suggesting that Fgfr1 provides a permissive environment for neural crest cell migration. Here we demonstrate localized down-regulation of the expression of the FGF responsive gene, Sprouty1 in the epithelium covering the presumptive second branchial arch of hypomorphic Fgfr1 mutants. This appears to result in a failure to establish an ectodermal signalling center expressing Fgf3 and Fgf15. We also studied differentiation of the ectoderm in the second branchial arch region. Development of the geniculate placode as well as the VIIth cranial ganglion is affected in Fgfr1 hypomorphs. Our results suggest that Fgfr1 is important for localized signalling in the pharyngeal ectoderm and consequently for normal tissue interactions in the developing second branchial arch.     

Dynamics of intracellular metabolites of glycolysis and TCA cycle during cell-cycle-related oscillation in Saccharomyces cerevisiae

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2- and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., alpha-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of beta-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and alpha-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors.     

Isolation, sequencing, and mutagenesis of the gene encoding NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) from Paracoccus denitrificans, in which GD-FALDH is essential for methylotrophic growth.

NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) of Paracoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.     

Methanol oxidation in a spontaneous mutant of Thiosphaera pantotropha with a methanol-positive phenotype is catalysed by a dye-linked ethanol dehydrogenase

Abstract A spontaneous Thiosphaera pantotropha mutant (Tp9002) that is able to grow on methanol has been isolated. With hybridization experiments it has been demonstrated that mxaF , the gene encoding the large subunit of methanol dehydrogenase, is absent from T. pantotropha . In Tp9002, a dye-linked enzyme activity was found with a substrate specificity similar to that of the dye-linked ethanol dehydrogenase from Pseudomonas aeruginosa . The N-terminus of a 26-kDa cytochrome c , exclusively synthesized in Tp9002, is homologous to the N-terminus of the electron acceptor of ethanol dehydrogenase. These results suggest that in Tp9002 a dye-linked ethanol dehydrogenase is responsible for methanol oxidation, using a 26-kDa cytochrome c as electron acceptor.     

Short-term metabolome dynamics and carbon, electron, and ATP balances in chemostat-grown Saccharomyces cerevisiae CEN.PK 113-7D following a glucose pulse

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O(2) uptake and CO(2) evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO(2) evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.     

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.     

Manipulation of insect gut microbiota towards the improvement of Bactrocera oleae artificial rearing

Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the main pest of olive trees (Olea europaea L.), causing major damages in olive crops. Improvement of mass rearing is a prerequisite for the successful development of large-scale sterile insect technique (SIT) applications. This can be achieved through the enrichment of artificial diets with gut bacteria isolates. We assessed the efficiency of three gut bacteria previously isolated from Ceratitis capitata (Wiedemann), and four isolated from B. oleae, as larval diet additives in both live and inactivated/dead forms. Our results showed that dead Enterobacter sp. AA26 increased pupal weight, whereas both live and dead cells increased pupal and adult production and reduced immature developmental time, indicating that its bacterial cells serve as a direct nutrient source. Live Providencia sp. AA31 improved pupal and adult production, enhanced male survival under stress conditions, and delayed immature development. Dead Providencia sp. AA31, however, did not affect production rates, indicating that live bacteria can colonize the insect gut and biosynthesize nutrients essential for larval development. Live and dead Bacillus sp. 139 increased pupal weight, accelerated immature development, and increased adult survival under stress. Moreover, live Bacillus sp. 139 improved adult production, indicating that Bacillus cells are a direct source of nutrients. Dead Serratia sp. 49 increased pupal and adult production and decreased male survival under stress conditions whereas live cells decreased insect production, indicating that the live strain is entomopathogenic, but its dead cells can be utilized as nutrient source. Klebsiella oxytoca, Enterobacter sp. 23, and Providencia sp. 22 decreased pupal and subsequent adult production and were harmful for B. oleae. Our findings indicate that dead Enterobacter sp. AA26 is the most promising bacterial isolate for the improvement of B. oleae mass rearing in support of future SIT or related population suppression programs.     

Activin A and Wnt-dependent specification of human definitive endoderm cells

Activin/Nodal and Wnt signaling are known to play important roles in the regional specification of endoderm. Here we have investigated the effect of the length of stimulation with Activin A plus Wnt3a on the development of hepatic and pancreatic progenitors from the definitive endoderm (DE) cells derived from human pluripotent stem cells (hPSC). We show that DE-cells derived from hPSC with 3 days high Activin A and Wnt3a treatment were able to differentiate further into both tested endodermal lineages. When prolonging the DE-induction protocol from 3 to 5 or 7 days, almost pure DE-marker positive cell populations were obtained. However, these cells had an impaired pancreatic differentiation capacity, while they still developed into hepatocyte-like cells. Further propagation of the DE-cells in the presence of Wnt3a and Activin A led to the complete loss of differentiation capacity into hepatic or pancreatic lineages. When Wnt3a was removed after 24 h from the initiation of the differentiation, the cells were able to differentiate into PDX1+/NKX6.1+ pancreatic progenitors even with longer DE induction time while efficiency of hepatic differentiation was lower. Our results suggest that both the length and the timing of Wnt3a treatment during DE induction are crucial for the final differentiation outcome. Although it is possible to derive apparently pure DE cells with prolonged Activin A/Wnt-stimulation, their progenitor capacity is restricted to a limited time window.