CPMG sequences with enhanced sensitivity to chemical exchange

Improved relaxation-compensated Carr–Purcell–Meiboom-Gill pulse sequences are reported for studying chemical exchange of backbone ¹⁵N nuclei. In contrast to the original methods [J. P. Loria, M. Rance, and A. G. Palmer, J. Am. Chem. Soc. 121, 2331–2332 (1999)], phenomenological relaxation rate constants obtained using the new sequences do not contain contributions from ¹H-¹H dipole-dipole interactions. Consequently, detection and quantification of chemical exchange processes are facilitated because the relaxation rate constant in the limit of fast pulsing can be obtained independently from conventional ¹⁵N spin relaxation measurements. The advantages of the experiments are demonstrated using basic pancreatic trypsin inhibitor.     

Spontaneous lipid vesicle fusion with electropermeabilized cells

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.     

Nod2 mediates susceptibility to Yersinia pseudotuberculosis in mice

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.     

Energy cost of walking and gait instability in healthy 65- and 80-yr-olds.

This study tested whether the lower economy of walking in healthy elderly subjects is due to greater gait instability. We compared the energy cost of walking and gait instability (assessed by stride to stride changes in the stride time) in octogenarians (G80, n = 10), 65-yr-olds (G65, n = 10), and young controls (G25, n = 10) walking on a treadmill at six different speeds. The energy cost of walking was higher for G80 than for G25 across the different walking speeds (P < 0.05). Stride time variability at preferred walking speed was significantly greater in G80 (2.31 +/- 0.68%) and G65 (1.93 +/- 0.39%) compared with G25 (1.40 +/- 0.30%; P < 0.05). There was no significant correlation between gait instability and energy cost of walking at preferred walking speed. These findings demonstrated greater energy expenditure in healthy elderly subjects while walking and increased gait instability. However, no relationship was noted between these two variables. The increase in energy cost is probably multifactorial, and our results suggest that gait instability is probably not the main contributing factor in this population. We thus concluded that other mechanisms, such as the energy expenditure associated with walking movements and related to mechanical work, or neuromuscular factors, are more likely involved in the higher cost of walking in elderly people.     

Biotinylated synthetic chemokines: their use for the development of nonradioactive whole-cell binding assays

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen.     

Selective use of TRAM in lipopolysaccharide (LPS) and lipoteichoic acid (LTA) induced NF-kappaB activation and cytokine production in primary human cells: TRAM is an adaptor for LPS and LTA signaling

TLR signal via Toll-IL-1R (TIR) homology domain-containing adaptor proteins. One of these adaptors, Toll-IL-1R domain-containing adaptor inducing IFN-beta-related adaptor molecule (TRAM), has been shown to be essential for TLR4 signaling in TRAM(-/-) mice and cell lines. Previously, we showed that MyD88 or Mal dominant-negative constructs did not inhibit LPS induction of cytokines in primary human M-CSF-derived macrophages. A possible explanation was redundancy of the adaptors during LPS signaling. TRAM is a suitable candidate to compensate for these adaptors. To investigate a potential role for TRAM in LPS signaling in human M-CSF-derived macrophages, we engineered an adenoviral construct expressing dominant-negative TRAM-C117H (AdTRAMdn). Synovial fibroblasts (SF) and human umbilical endothelial cells (HUVECs) were used as a nonmyeloid comparison. AdTRAMdn inhibited LPS-induced signaling in SFs and HUVECs, reducing NF-kappaB activation and cytokine production, but did not inhibit LPS signaling in M-CSF-derived human macrophages. Further investigation of other TLR ligands showed that AdTRAMdn was also able to inhibit signaling initiated by lipoteichoic acid, a TLR2 ligand, in SFs and HUVECs and lipoteichoic acid and macrophage-activating lipopeptide 2 signaling was also inhibited in TRAM(-/-) murine embryonic fibroblasts. We conclude that TRAM is an adaptor protein for both TLR4 and TLR2/6 signaling in SFs, HUVECs, and murine embryonic fibroblasts, but cannot demonstrate a role in human macrophages.     

A trophic pathway from biogenic methane supports fish biomass in a temperate lake ecosystem

Although some primary consumers such as chironomid larvae are known to exploit methane‐derived carbon via microbial consortia within aquatic food webs, few studies have traced the onward transfer of such carbon to their predators. The ruffe Gymnocephalus cernuus is a widespread benthivorous fish which feeds predominantly on chironomid larvae and is well adapted for foraging at lower depths than other percids. Therefore, any transfer of methanogenic carbon to higher trophic levels might be particularly evident in ruffe. We sampled ruffe and chironomid larvae from the littoral, sub‐littoral and profundal areas of Jyväsjärvi, Finland, a lake which has previously been shown to contain chironomid larvae exhibiting the very low stable carbon isotope ratios indicative of methane exploitation. A combination of fish gut content examination and stable isotope analysis was used to determine trophic linkages between fish and their putative prey. Irrespective of the depth from which the ruffe were caught, their diet was dominated by chironomids and pupae although the proportions of taxa changed. Zooplankton made a negligible contribution to ruffe diet. A progressive decrease in δ¹³C and δ¹⁵N values with increasing water column depth was observed for both chironomid larvae and ruffe, but not for other species of benthivorous fish. Furthermore, ruffe feeding at greater depths were significantly larger than those feeding in the littoral, suggesting an ontogenetic shift in habitat use, rather than diet, as chironomids remained the predominant prey item. The outputs from isotope mixing models suggested that the incorporation of methane‐derived carbon to larval chironomid biomass through feeding on methanotrophic bacteria increased at greater depth, varying from 0% in the littoral to 28% in the profundal. Using these outputs and the proportions of littoral, sub‐littoral or profundal chironomids contributing to ruffe biomass, we estimated that 17% of ruffe biomass in this lake was ultimately derived from chemoautotrophic sources. Methanogenic carbon thus supports considerable production of higher trophic levels in lakes.     

Species Diversity,Phylogeny and Large Scale Biogeographic Patterns of the Genus Padina (Phaeophyceae,Dictyotales)

The brown algal genus Padina (Dictyotales, Phaeophyceae) is distributed worldwide in tropical and temperate seas. Global species diversity and distribution ranges, however, remain largely unknown. Species‐level diversity was reassessed using DNA‐based, algorithmic species delineation techniques based on cox3 and rbcL sequence data from 221 specimens collected worldwide. This resulted in estimates ranging from 39 to 61 putative species (ESUs), depending on the technique as well as the locus. We discuss the merits, potential pitfalls, and evolutionary and biogeographic significance of algorithmic species delineation. We unveil patterns whereby ESUs are in all but one case restricted to either the Atlantic or Indo‐Pacific Ocean. Within ocean basins we find evidence for the vast majority of ESUs to be confined to a single marine realm. Exceptions, whereby ESUs span up to three realms, are located in the Indo‐Pacific Ocean. Patterns of range‐restricted species likely arise by repeated founder events and subsequent peripatric speciation, hypothesized to dominate speciation mechanisms for coastal marine organisms in the Indo‐Pacific. Using a three‐gene (cox3, psaA and rbcL), relaxed molecular clock phylogenetic analysis we estimated divergence times, providing a historical framework to interpret biogeographic patterns.