Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.     

Mapping of Bacterial Biofilm Local Mechanics by Magnetic Microparticle Actuation

Most bacteria live in the form of adherent communities forming three-dimensional material anchored to artificial or biological surfaces, with profound impact on many human activities. Biofilms are recognized as complex systems but their physical properties have been mainly studied from a macroscopic perspective. To determine biofilm local mechanical properties, reveal their potential heterogeneity, and investigate their relation to molecular traits, we have developed a seemingly new microrheology approach based on magnetic particle infiltration in growing biofilms. Using magnetic tweezers, we achieved what was, to our knowledge, the first three-dimensional mapping of the viscoelastic parameters on biofilms formed by the bacterium Escherichia coli. We demonstrate that its mechanical profile may exhibit elastic compliance values spread over three orders of magnitude in a given biofilm. We also prove that heterogeneity strongly depends on external conditions such as growth shear stress. Using strains genetically engineered to produce well-characterized cell surface adhesins, we show that the mechanical profile of biofilm is exquisitely sensitive to the expression of different surface appendages such as F pilus or curli. These results provide a quantitative view of local mechanical properties within intact biofilms and open up an additional avenue for elucidating the emergence and fate of the different microenvironments within these living materials.     

Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.     

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.     

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling^1,2, while LRRK2 is implicated in the pathogenesis of Parkinson''s disease^3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with ³²P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (³²P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.     

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes

Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone marrow, their characteristics differ on the compartment from which they are derived. In this work, we investigated the contribution of phagocytosis for tumor necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently, depending on their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors was sufficient for a maximal production of TNF and interleukin-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating the different actors of signalization, we found that p38 kinase and phosphatidylinositol 3-kinase were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α(5)β(1)-integrin significantly decreased TNF production in response to HKSA in all three cell types. Finally, using mononuclear phagocytes from NOD2 knockout mice, we observed that TNF production in response to HKSA was dependent on NOD2 for monocytes and peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways. The influence of the compartments on cell properties and behavior should be taken into account, to better understand cell physiology and host-pathogen interaction, and to define efficient strategies to fight infection.     

Adherence modifies the regulation of gene expression induced by interleukin-10

Interleukin-10 (IL-10) is a well known anti-inflammatory cytokine. However, we previously showed that it could present pro-inflammatory properties on human monocytes in the absence of adherence. In the present study, using macroarray technology, we analyzed the effects of IL-10 and adherence on the expression of 1050 genes in human monocytes cultured for 3 hours on plastic or Teflon(R) (to avoid adherence). Adherence alone induced specifically the expression of 12 genes and repressed that of 25 genes. In adherent monocytes, IL-10 induced the expression of 21 genes and repressed that of 50 genes. In non-adherent monocytes, IL-10 induced the expression of 45 genes and repressed that of 67. Only 3 common genes were induced while 35 common genes were repressed by IL-10 in the two culture conditions. Interestingly, we showed that IL-10 modulated conversely on Teflon(R) and plastic the expression of 16 genes, of which SOCS molecules, coproporphyrinogen oxidase, matrix metalloproteinases and complement receptor-1 (CD35). This study demonstrates that adherence has profound modulatory effects on the properties and the signaling induced by IL-10. The discovery that IL-10 can inhibit the production of coproporphyrinogen oxidase (an enzyme involved in the synthesis of heme) may shed some lights on the mechanisms of anaemia induced by IL-10. Furthermore, the inhibition of the expression of SOCS1 by IL-10 in the absence of adherence, may explain its priming effects on a subsequent LPS stimulation that we previously described.     

Anaerobic/Aerobic Composting of Soil Contaminated with 2,4,6-Trinitrotoluene

A loam soil from Pennsylvania without a history of exposure to explosives was incubated with 5 g kg^-1 of ¹⁵N-labeled 2,4,6-trinitrotoluene (TNT) and 200 μCi kg^-1 of ¹⁴C-TNT for 3 days and then amended with compost at a 1:2 soil to compost ratio. The compost was prepared by mixing 40% alfalfa hay, 40% grass hay, 10% spent mushroom compost, and 10% municipal biosolids. The mixture of soil and compost was inoculated with methanogens from cattle manure, amended with glucose and starch, and incubated for 37 days under anaerobic conditions. The anaerobic incubation was followed by 26 days of forced aerobic incubation. At the end of the aerobic phase, most of the radioactivity was associated with organic matter; only 8.7% could be extracted with water and methanol, but no TNT was present in the extracts as determined by high-performance liquid chromatography. The unextractable radioactivity was associated with humic acid (40.0±1.0%), fulvic acid (14.3±1.4%), and humin (28.2±0.5%). Radioactive materials associated with humic acid and humin were analyzed by solid-state ¹⁵N-nuclear magnetic resonance (NMR) spectrometry. The NMR spectra indicated that nitro groups of TNT had been reduced to amino groups thatwere subsequently involved in the formation of covalent bonds with soil organic matter.     

Isotopic labelling of photosystem II in Thermosynechococcus elongatus

This report describes a protocol to incorporate isotopically labelled aromatic amino acids into the proteins of the thermophilic cyanobacterium Thermosynechoccus elongatus. By using the EPR signal of the two redox active tyrosines of Photosystem II, Tyr(D)(*) and Tyr(Z)(*), as spectroscopic probes it is shown that labelled tyrosines can be incorporated with a high yield in this cyanobacterium. The production of a fully (13)C- or (2)H-labelled enzyme is also described.