Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM alphaW62A,betaW120A (DM(W62A/W120A)) double mutant was expressed in HLA-DR(+) HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.     

Stoichiometry of aerobic mineralization (O/C) of aquatic macrophytes leachate from a tropical lagoon (São Paulo –Brazil)

The temporal variation of stoichiometry between consumed oxygen and oxidized carbon was investigated for the aerobic mineralization of leachates from aquatic macrophytes. Seven species of aquatic plants, viz. Cabomba piauhyensis, Cyperus giganteus, Egeria najas, Eichhornia azurea, Salvinia auriculata, Scirpus cubensisand Utricularia breviscapa, were collected from Òleo lagoon located in the floodplain of Mogi-Guacu river (São Paulo State, Brazil). After being collected, the plants were washed, oven-dried and triturated. In order to obtain the leachate, the fragments were submitted to an aqueous extraction (cold). Mineralization chambers were incubated at 20 °C containing leachates dissolved in water samples from Òleo lagoon to a final concentration of ca. 200 mg l^–1on carbon basis. The chambers were maintained under aerobic conditions; the concentrations of the organic carbon (particulate and dissolved) and the dissolved oxygen were measured during approximately 80 days. Elemental analysis of the detritus and the concentrations of the remaining material (DOC and POC) were used to determine the amounts of mineralized organic carbon. The data were analyzed with first-order kinetics models, from which the daily rates of consumption (carbon and oxygen) and the stoichiometry (O/C) were determined. In the early phase of mineralization the O/C rates increased before reaching a maximum, after which they tended to decrease. For the mineralization of leachates from C. giganteus, S. auriculata and U. breviscapa, the decrease was relatively slow. For all substrata the initial values were smaller than 1, and ranged from 0.42 (S. cubensis) to 0.81 (C. piauhyensis). The maximum values were within the range from 0.58 (U. breviscapa) to 23.1 (E. najas) and at their highest 26th (C. piauhyensis) and 106th (C. giganteus) days. These variations are believed to be associated with the chemical composition of the leachates, with their transformations and alterations of metabolic pathways involved in the mineralization.     

The source of gibberellins in the parthenocarpic development of ovaries on topped pea plants

The role and source of gibberellins (GAs) involved in the development of parthenocarpic fruits of Pisum sativum L. has been investigated. Gibberellins applied to the leaf adjacent to an emasculated ovary induced parthenocarpic fruit development on intact plants. The application of gibberellic acid (GA₃) had to be done within 1 d of anthesis to be fully effective and the response was concentration-dependent. Gibberellin A₁ and GA₃ worked equally well and GA₂₀ was less efficient. [³H]Gibberellin A₁ applied to the leaf accumulated in the ovary and the accumulation was related to the growth response. These experiments show that GA applied to the leaf in high enough concentration is translocated to the ovary. Emasculated ovaries on decapitated pea plants develop without application of growth hormones. When [³H] GA₁ was applied to the leaf adjacent to the ovary a substantial amount of radioactivity accumulated in the growing shoot of intact plants. In decapitated plants, however, this radioactivity was mainly found in the ovary. There it caused growth proportional to the accumulation of CA₁. Application of LAB 150978, an inhibitor of GA biosynthesis, to decapitated plants inhibited parthenocarpic fruit development and this inhibition was counteracted by the application of GA₃ (either to the fruit, or the leaf adjacent to the ovary, or through the lower cut end of the stem). All evidence taken together supports the view that parthenocarpic pea fruit development on topped plants depends on the import of gibberellins or their precursors, probably from the vegetative aerial parts of the plant.Abbreviations FW flesh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

Long-term consumption of a diet with moderate medium chain triglyceride content does not inhibit the activity of enzymes involved in hepatic lipogenesis in the rat

The activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase were lower (-25 to -60%) in liver of rats fed during 45 days with a moderate long-chain triglycerides (LCT) content diet (32% of metabolizable energy, ME), than in control rats fed with a low fat diet (LCT, 10% of ME). However, the fall in malic enzyme activity was not significant. In contrast, these activities were higher (+40 to +160%) in rats fed with a diet with a moderate medium-chain triglycerides (MCT) content (32% of ME), than in control rats. Nevertheless, the increase in activity of malic enzyme and ATP-citrate lyase was more important. Contrary to LCTs, MCTs had no inhibitory effect on the activity of enzymes involved in hepatic lipogenesis.     

Next-generation sequencing for identifying new genes in rare genetic diseases: many challenges and a pinch of luck

A report on the European Society of Human Genetics conference, held in Paris, France, June 8-11, 2013.     

Representation of Ecosystem Services by Terrestrial Protected Areas: Chile as a Case Study

Protected areas are increasingly considered to play a key role in the global maintenance of ecosystem processes and the ecosystem services they provide. It is thus vital to assess the extent to which existing protected area systems represent those services. Here, for the first time, we document the effectiveness of the current Chilean protected area system and its planned extensions in representing both ecosystem services (plant productivity, carbon storage and agricultural production) and biodiversity. Additionally, we evaluate the effectiveness of protected areas based on their respective management objectives. Our results show that existing protected areas in Chile do not contain an unusually high proportion of carbon storage (14.9%), agricultural production (0.2%) or biodiversity (11.8%), and also represent a low level of plant productivity (Normalized Difference Vegetation Index of 0.38). Proposed additional priority sites enhance the representation of ecosystem services and biodiversity, but not sufficiently to attain levels of representation higher than would be expected for their area of coverage. Moreover, when the species groups were assessed separately, amphibians was the only one well represented. Suggested priority sites for biodiversity conservation, without formal protection yet, was the only protected area category that over-represents carbon storage, agricultural production and biodiversity. The low representation of ecosystem services and species’ distribution ranges by the current protected area system is because these protected areas are heavily biased toward southern Chile, and contain large extents of ice and bare rock. The designation and management of proposed priority sites needs to be addressed in order to increase the representation of ecosystem services within the Chilean protected area system.     

Specific Midgut Region Controlling the Symbiont Population in an Insect-Microbe Gut Symbiotic Association

Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host''s control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug Riptortus pedestris harbors a betaproteobacterial extracellular symbiont of the genus Burkholderia in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors Burkholderia symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the Burkholderia symbiont but not the cultured Burkholderia and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of R. pedestris that plays a critical role in controlling the population of the Burkholderia gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.     

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.