IFN-gamma enhances production of nitric oxide from macrophages via a mechanism that depends on nucleotide oligomerization domain-2

Pattern recognition receptors are central to the responsiveness of various eukaryotic cell types when they encounter pathogen-associated molecular patterns. IFN-gamma is a cytokine that is elevated in humans and other animals with bacterial infection and enhances the LPS-induced production of antibacterial mediators by macrophages. Mice lacking the pattern recognition receptor, TLR4, respond very poorly to stimulation by LPS, but administration of IFN-gamma has been described as restoring apparent sensitivity to this stimulatory ligand. In this study, we show that IFN-gamma primes murine macrophages stimulated by crude LPS preparations to produce the antibacterial mediator NO, a proportion of which is independent of TLRs 2 and 4. This response is lost in tlr4-/- IFN-gamma-primed murine macrophages when the LPS preparation is highly purified. NO is also induced if chemically synthesized muramyl dipeptide, an intermediate in the biosynthesis of peptidoglycan, is used to stimulate macrophages primed with IFN-gamma. This is absolutely dependent on the presence of a functional nucleotide oligomerization domain-2 (NOD-2) protein. IFN-gamma increases NOD-2 expression and dissociates this protein from the actin cytoskeleton within the cell. IFN-gamma priming of macrophages therefore reveals a key proinflammatory role for NOD-2. This study also shows that the effect of IFN-gamma in restoring inflammatory responses to gram-negative bacteria or bacterial products in mice with defective TLR4 signaling is likely to be due to a response to peptidoglycan, not LPS.     

The crystal structure of TrpD, a metabolic enzyme essential for lung colonization by Mycobacterium tuberculosis, in complex with its substrate phosphoribosylpyrophosphate

Mycobacterium tuberculosis, the cause of tuberculosis, presents a major threat to human health worldwide. Biosynthetic enzymes that are essential for the survival of the bacterium, especially in activated macrophages, are important potential drug targets. Although the tryptophan biosynthesis pathway is thought to be non-essential for many pathogens, this appears not to be the case for M.tuberculosis, where a trpD gene knockout fails to cause disease in mice. We therefore chose the product of the trpD gene, anthranilate phosphoribosyltransferase, which catalyses the second step in tryptophan biosynthesis, for structural analysis. The structure of TrpD from M.tuberculosis was solved by X-ray crystallography, at 1.9 A resolution for the native enzyme (R = 0.191, Rfree = 0.230) and at 2.3 A resolution for the complex with its substrate phosphoribosylpyrophosphate (PRPP) and Mg2+ (R = 0.194, Rfree = 0.255). The enzyme is folded into two domains, separated by a hinge region. PRPP binds in the C-terminal domain, together with a pair of Mg ions. In the substrate complex, two flexible loops change conformation compared with the apo protein, to close over the PRPP and to complete an extensive network of hydrogen-bonded interactions. A nearby pocket, adjacent to the hinge region, is postulated by in silico docking as the binding site for anthranilate. A bound molecule of benzamidine, which was essential for crystallization and is also found in the hinge region, appears to reduce flexibility between the two domains.     

Growth and photosynthetic responses to salinity in an extreme halophyte, Sarcocornia fruticosa

Sarcocornia fruticosa (L.) A.J. Scott is found in coastal marshes of south-west Spain, growing under a very wide range of interstitial soil salinity from 10 m M up to nearly 1000 m M . A glasshouse experiment was designed to investigate the effect of this range of salinities on the morphology and the photosynthetic apparatus of S. fruticosa by measuring growth rate, photosynthetic and non-photosynthetic area, atrophy of distal branch ends, water status, chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. The long-term effects of salinity on the growth of S. fruticosa were mainly determined by the extent of photosynthetic area rather than the variations in net photosynthetic rate. Photosynthetic area was reduced at 1030 m M as a result of a decrease in the length of the photosynthetic portions. This was induced by fewer internodes and, at salinities lower than 510 m M , smaller internode diameter. Net photosynthetic rate increased as the quantum efficiency of photosystem II decreased in the different salinity treatments, which means that the plant could be increasing photorespiration and/or using cyclic electron transport as additional photoprotective mechanisms. The recorded drop in net photosynthetic rate at higher salinities appeared to be due to a reduction in stomatal conductance. The results indicate that S. fruticosa is capable of tolerating very high and continued exposure to salt, showing its greatest growth rate at 510 m M NaCl.     

Expression and inhibition of aquaporins in germinating Arabidopsis seeds

Extensive and kinetically well-defined water exchanges occur during germination of seeds. A putative role for aquaporins in this process was investigated in Arabidopsis. Macro-arrays carrying aquaporin gene-specific tags and antibodies raised against aquaporin subclasses revealed two distinct aquaporin expression programs between dry seeds and young seedlings. High expression levels of a restricted number of tonoplast intrinsic protein (TIP) isoforms (TIP3;1 and/or TIP3;2, and TIP5;1) together with a low expression of all 13 plasma membrane aquaporin (PIP) isoforms was observed in dry and germinating materials. In contrast, prevalent expression of aquaporins of the TIP1, TIP2 and PIP subgroups was induced during seedling establishment. Mercury (5 microM HgCl(2)), a general blocker of aquaporins in various organisms, reduced the speed of seed germination and induced a true delay in maternal seed coat (testa) rupture and radicle emergence, by 8-9 and 25-30 h, respectively. Most importantly, mercury did not alter seed lot homogeneity nor the seed germination developmental sequence, and its effects were largely reversed by addition of 2 mM dithiothreitol, suggesting that these effects were primarily due to oxidation of cell components, possibly aquaporins, without irreversible alteration of cell integrity. Measurements of water uptake in control and mercury-treated seeds suggested that aquaporin functions are not involved in early seed imbibition (phase I) but would rather be associated with a delayed initiation of phase III, i.e. water uptake accompanying expansion and growth of the embryo. A possible role for aquaporins in germinating seeds and more generally in plant tissue growth is discussed.     

Disruption of the Arabidopsis SMC4 gene, AtCAP-C, compromises gametogenesis and embryogenesis

In higher eukaryotes, the condensin complex is a multisubunit apparatus that plays a pivotal role in the coordinated condensation of chromatin during mitosis. The catalytic subunits, CAP-E and CAP-C, members of the SMC family of ATPases, form a heterodimer, the activity of which is controlled by the non-SMC subunits CAP-D2, CAP-G and CAP-H. Here, we report the characterization of a T-DNA insertion mutant of the Arabidopsis CAP-C gene. Analysis of the progeny of selfed heterozygotes revealed that the homozygous null genotype is embryo lethal, with arrest occurring at or before the globular stage of development. Patterning defects associated with altered planes of cytokinesis were found in both the embryo and the suspensor. Crosses of heterozygotes with wild type plants revealed both male and female gametophytic defects. Stretched chromatin was observed between segregating mitotic chromosomes in pollen produced by selfed heterozygotes. Additionally, some plants heterozygous for the T-DNA insertion exhibited loss of apical dominance and mild fasciation, indicating a semi-dominant effect of the mutation. These results reveal a critical role for AtCAP-C during cell division and, unlike our previous studies on the AtCAP-E genes, suggest that no redundant factors for AtCAP-C exist in the Arabidopsis genome.     

The first gene-based map of Lupinus angustifolius L.-location of domestication genes and conserved synteny with Medicago truncatula

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorised users.     

Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor

Preterm delivery is the leading cause of perinatal mortality and morbidity. Current tocolytics target myometrial contractions, a late step in the labor cascade. Identifying earlier events in parturition may lead to more effective therapeutic strategies. We hypothesized that inflammatory events in decidua (the maternal-fetal interface), characterized by leucocyte infiltration, are an early event during term and preterm labor (PTL). Leucocyte abundance in decidua of human pregnancies was quantified following term labor and PTL (idiopathic and infection associated), in conjunction with investigation of temporal inflammatory events in rat uterus during the perilabor period and in PTL induced by mifepristone. In human decidua, macrophage numbers were 4-fold higher in term labor (P < 0.01) and 2.5-fold higher in non-infection-associated PTL (P < 0.05) than in term nonlaboring samples. Neutrophil abundance was unchanged with labor but elevated in PTL with infection (5- to 53-fold increase; P < 0.01). T and NK cells were more abundant in idiopathic PTL than TL (P < 0.05). In rat, decidual macrophage infiltration increased 4.5-fold 12 h prior to labor and remained elevated during labor and early postpartum (P < 0.01). Decidual infiltration preceded that of the myometrium and was 4-fold higher (P < 0.01). In rat PTL, decidual macrophage numbers were also elevated (P < 0.01) and exceeded those of the myometrium (P < 0.05). These studies show for the first time that leucocytes infiltrate decidua during labor at term and preterm, supporting a role for leucocyte-derived inflammatory mediators in decidual activation. In the rat, this occurred prior to labor, suggesting it is an early event during parturition and thus a potential target for intervention.     

Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans

Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.     

Identification of proteins susceptible to thiol oxidation in endothelial cells exposed to hypochlorous acid and N-chloramines

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis.     

Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.