Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1

The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes. The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation. The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753. The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.     

Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila

Summary The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism.The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently. Therefore the mechanism of resistance to aminopterin in Drosophila may be different from those described for methotrexate in mammalian cultured cells, as indicated by the results obtained for D. ananassae.     

Study of the effect of 2 inhibitors of nucleotide synthesis, aminopterin and fluorodeoxyuridine, on wild type strains and vestigial mutants in Drosophila melanogaster

Two inhibitors of nucleotide metabolism, aminopterin and FUdR, were tested on a wild type strain, on two mutant strains: vg and vgnp, and on a vg strain with the wild type genetic background. Without inhibitors, a lengthening of the developing time was observed for the mutant strains compared to the wild type. With aminopterin, larval mortality and lengthening of developing time are significantly higher in the wild type than in the mutant strains. Mutant strains seemed to be resistant to low concentrations of FUdR. The hypothesis of a perturbed pyrimidine metabolism in the mutants seems to be confirmed.     

Effect of flurbiprofen and acetylsalicylic acid on the ultrastructure of blood platelets

Flurbiprofene or acetylsalicylic acid did not change the structure of inactivated platelets. With flurbiprofene 50% aggregation inhibition was obtained at 10(-6) to 10(-5) M concentrations. To obtain the same result with acetylsalicylic acid, 10(-4) to 10(-3) M concentrations were necessary. With both agents, shape change was inhibited. The platelets in the small aggregates did not have the normal stretched dumb-bell shape but remained globulous and emitted a broad pseudopode containing normally-repolymerized microtubules.     

Impact of interpopulation divergence on additive and dominance variance in hybrid populations

We present a theoretical proof that the ratio of the dominance vs. the additive variance decreases with increasing genetic divergence between two populations. While the dominance variance is the major component of the variance due to specific combining ability (sigma(SCA)(2)), the additive variance is the major component of the variance due to general combining ability (sigma(GCA)(2)). Therefore, we conclude that interpopulation improvement becomes more efficient with divergent than with genetically similar heterotic groups, because performance of superior hybrids can be predicted on the basis of general combining ability effects.     

Plasmonic Enhancement of Fluorescence and Raman Scattering by Metal Nanotips

We report modifications to the optical properties of fluorophores in the vicinity of noble metal nanotips. The fluorescence from small clusters of quantum dots has been imaged using an apertureless scanning near-field optical microscope. When a sharp gold tip is brought close to the sample surface, a strong distance-dependent enhancement of the quantum dot fluorescence is observed, leading to a simultaneous increase in optical resolution. These results are consistent with simulations of the electric field and fluorescence enhancement near plasmonic nanostructures. Highly ordered periodic arrays of silver nanotips have been fabricated by nanosphere lithography. Using fluorescence lifetime imaging microscopy, we have created high-resolution spatial maps of the lifetime components of vicinal fluorophores; these show an order of magnitude increase in decay rate from a localized volume around the nanotips, resulting in a commensurate enhancement in the fluorescence emission intensity. Spatial maps of the Raman scattering signal from molecules on the nanotips shows an enhancement of more than five orders of magnitude.     

Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-microl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 +/- 0.7) x 10(3) to (2.9 +/- 0.3) x 10(5) copies of nagAc-like dioxygenase genes per microg of DNA extracted from sediment samples. These values corresponded to (1.2 +/- 0.6) x 10(5) to (5.4 +/- 0.4) x 10(7) copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.     

Beach morphology and food web structure: comparison of an eroding and an accreting sandy shore in the North Sea

Food web components and inorganic nutrients were studied on two sandy shores of the adjacent barrier islands of Sylt and R?m? in the North Sea, differing in morphodynamics. Implications of high and low wave energy on the food web structure were assessed. The Sylt shore represents a dynamic intermediate beach type, while the R?m? shore is morphologically stable and dissipative. On the steep-profiled, coarse-grained Sylt shore, strong hydrodynamics resulted in erosion and high fluxes of organic material through the beach, but prevented any storage of food sources. In contrast, the flat-profiled, fine-grained R?m? shore, with low wave energy and accretion, accumulated organic carbon from surf waters. At Sylt, oxic nutrient regeneration prevailed, while anoxic mineralization was more important at R?m?. Macrofauna on the Sylt shore was impoverished compared with the community at R?m?. Correspondingly, abundances of epibenthic predators such as shrimps, crabs, fish, and shorebirds were also lower at Sylt. Meiofauna was abundant on both shores, but differed in taxonomic composition. Several major taxa were represented in fairly equal proportions of individual numbers on the well-oxygenated Sylt shore, while nematodes strongly dominated the assemblage at R?m?. Thus, on cold-temperate, highly dynamic intermediate shores with high wave energy and subject to erosion, the "small food web" dominates. Organisms are agile and quickly exploit fresh organic material. Larger organisms and nematodes abound under stable, dissipative and accreting shore conditions, where some food materials may accumulate and zoomass builds up to support numerous visitors from higher trophic levels. Electronic Publication     

Ceramide-induced TCR up-regulation

The TCR is a constitutively recycling receptor meaning that a constant fraction of TCR from the plasma membrane is transported inside the cell at the same time as a constant fraction of TCR from the intracellular pool is transported to the plasma membrane. TCR recycling is affected by protein kinase C activity. Thus, an increase in protein kinase C activity affects TCR recycling kinetics leading to a new TCR equilibrium with a reduced level of TCR expressed at the T cell surface. Down-regulation of TCR expression compromises T cell activation. Conversely, TCR up-regulation is expected to increase T cell responsiveness. The purpose of this study was to identify and characterize potential pathways for TCR up-regulation. We found that ceramide affected TCR recycling dynamics and induced TCR up-regulation in a concentration- and time-dependent manner. Experiments applying phosphatase inhibitors indicated that ceramide-induced TCR up-regulation was most probably mediated by serine/threonine protein phosphatase 2A. Analyses of T cell variants demonstrated that TCR up-regulation was dependent on the presence of an intact CD3gamma L-based motif and thus acted on TCR engaged in the recycling pathway. Finally, we showed that TCR up-regulation probably plays a physiological role by increasing T cell responsiveness. Thus, by affecting the TCR recycling kinetics, T cells have the potential both to up- and down-regulate TCR expression and thereby adjust T cell responsiveness.