Functional Diversity of Plant–Pollinator Interaction Webs Enhances the Persistence of Plant Communities

Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm species. Thus, pollinators provide an essential ecosystem service to humankind. However, the impact of human-induced biodiversity loss on the functioning of plant–pollinator interactions has not been tested experimentally. To understand how plant communities respond to diversity changes in their pollinating fauna, we manipulated the functional diversity of both plants and pollinators under natural conditions. Increasing the functional diversity of both plants and pollinators led to the recruitment of more diverse plant communities. After two years the plant communities pollinated by the most functionally diverse pollinator assemblage contained about 50% more plant species than did plant communities pollinated by less-diverse pollinator assemblages. Moreover, the positive effect of functional diversity was explained by a complementarity between functional groups of pollinators and plants. Thus, the functional diversity of pollination networks may be critical to ecosystem sustainability.     

Dendritic cell differentiation and immune tolerance to insulin-related peptides in Igf2-deficient mice

There is some evidence that insulin-like growth factor 2 (IGF-2) may intervene in the control of T cell differentiation. To further study the immunoregulatory function of this growth factor, we analyzed the immune system of Igf2-/- mice. Phenotypically, some immunological parameters such as lymphoid organ morphology and cellularity were unaltered in Igf2-/- mice, but an increase of CD8+ cells and a decrease of B220+ cells were observed in spleen. In vitro, the development of bone marrow-derived dendritic cells was affected by the absence of Igf2 expression. After maturation, a higher percentage of immature dendritic cells was observed in Igf2-/- population, together with a secondary decrease in allogenic T cell proliferation. Activation of T cells was also affected by the lack of expression of this growth factor. The profile of B cell response in mutant mice immunized with IGF-2 evidenced a T-dependent profile of anti-IGF-2 Abs that was absent in Igf2+/+ mice. The influence of IGF-2 upon tolerance to insulin was also assessed in this model, and this showed that IGF-2 also intervenes in tolerance to insulin. The presence of a T-dependent response in Igf2-deficient mice should allow cloning of specific "forbidden" T CD4+ lymphocytes directed against IGF-2, as well as further investigation of their possible pathogenic properties against insulin family.     

Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4A resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall.     

Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.     

A Naturally Occurring Variant in Human TLR9, P99L, Is Associated with Loss of CpG Oligonucleotide Responsiveness

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.     

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.     

Design of a data model for developing laboratory information management and analysis systems for protein production

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.     

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.     

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: Evidence for a signalling role of docosahexaenoic acid

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport.We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced d-[³H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with α-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[³H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on d-[³H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions.These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.     

Could a defective epithelial sodium channel lead to bronchiectasis

Background

Bronchiectasis is defined as a permanent dilation of the airways arising from chronic bronchial inflammation/infection. In 50% of cases, no etiology can be identified. Recently, the role of the epithelial sodium channel ENaC has been pointed out in the pathophysiology of cystic fibrosis, a disease due to mutations in the CFTR gene and causing bronchiectasis in the airways. Moreover, it was found that transgenic mice overexpressing ENaCβ present cystic fibrosis-like lung disease symptoms. Our aim was to evaluate if a defective ENaC protein could be involved in the development of bronchiectasis.

Methods

We extensively analysed ENaCβ and γ genes in 55 patients with idiopathic bronchiectasis and without two mutations in the coding regions of CFTR. Thirty-eight patients presented functional abnormalities suggesting impaired sodium transport (abnormal sweat chloride concentration or nasal potential difference measurement), and 17 had no such evidence.

Results

Sequencing of the exons and flanking introns of the ENaCβ and γ gene identified five different amino-acid changes (p.Ser82Cys, p.Pro369Thr, p.Asn288Ser in ENaCβ ; and p.Gly183Ser, p.Glu197Lys in ENaCγ) in heterozygous state in 8 patients. The p.Ser82Cys amino-acid change was found in 3 unrelated patients who were also heterozygous for a CFTR mutation or variant (1 p.F508del, 1 IVS8-5T, and 1 IVS8-5T:1716G>A (p.E528E)). The other mutations were found in patients without CFTR mutation, the p.Glu197Lys mutation in 2 patients and the other variants in single patients. Among the 8 patients bearing an ENaC mutation, 5 had functional abnormalities suggesting impaired sodium transport.

Conclusion

Our results suggest that several variants in ENaCβ and γ genes might be deleterious for ENaC function and lead to bronchiectasis, especially in patients who are trans-heterozygotes for ENaCβ/CFTR mutations or variants.