Gene expression profiling studies on Caenorhabditis elegans dystrophin mutants dys-1(cx-35) and dys-1(cx18)

The Caenorhabditis elegans genome contains a single dystrophin/utrophin orthologue, dys-1. Point mutations in this gene, dys-1(cx35) and dys-1(cx18), result in truncated proteins. Such mutants offer potentially valuable worm models of human Duchenne muscular dystrophy. We have used microarrays to examine genes expressed differentially between wild-type C. elegans and dys-1 mutants. We found 106 genes (115 probe sets) to be differentially expressed when the two mutants are compared to wild-type worms, 49 of which have been assigned to six functional categories. The main categories of regulated genes in C. elegans are genes encoding intracellular signalling, cell-cell communication, cell-surface, and extracellular matrix proteins; genes in these same categories have been shown by others to be differentially expressed in muscle biopsies of muscular dystrophy patients. The C. elegans model may serve as a convenient vehicle for future genetic and chemical screens to search for new drug targets.     

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.     

Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.     

A large T cell invagination with CD2 enrichment resets receptor engagement in the immunological synapse

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.     

Environmental Profile of Brazilian Green Coffee (6 pp)

Goal, Scope and Background

Brazil is the world's biggest producer of coffee beans with approx. a 30% market share. Depending on climate conditions, approx. 30 million bags of coffee beans are exported annually from Brazil, while domestic consumption is around 10 million bags, which makes Brazil the world's third largest coffee-consuming country. Therefore, the goal of this paper is to present the LCA of green coffee produced in Brazil for the reference crops 2001/02 and 2002/03 in order to generate detailed production inventory data as well as to identify the potential environmental impacts of its tillage in order to realize how to reduce those impacts and increase the environmental sustainability of this product. Only the inputs and outputs relative to the coffee tillage were considered. The production of fertilizers, correctives and pesticides were not included in the boundary, but only their amounts. The functional unit selected for this study was 1,000 kg of green coffee destined for exportation.

Methods

The LCI was performed according to the ISO 14040 standard series. All information considered in this study (use of water, fossil based energy, fertilizers and chemicals) were taken up in in-depth data collection and evaluation by questionnaires applied on a farm level and/or received by mail. Four Brazilian coffee producer regions were evaluated: Cerrado Mineiro, South of Minas Gerais State, the Marília and Alta Mogiana regions in São Paulo State. These regions have the following geographic coordinates: 44 to 50° W longitude and 18 to 24° S latitude. The data refer to a production of 420,000 coffee bean bags and a productive area of approx. 14,300 ha. The varieties of coffee beans considered in this study were Mundo Novo, Catuaí (yellow and red), Icatu (yellow and red), Catucaí (yellow and red) and Obatã. Farm specific data along with agricultural production data have been combined to elaborate a coffee cultivation inventory, which will be applied in an emissions estimation.

Results and Conclusion

The production of 1,000 kg of green coffee in Brazil requires approx. 11,400 kg of water, 94 kg of diesel, 270 kg of fertilizers as NPK, 900 kg of total fertilizers, 620 kg of correctives, 10 kg of pesticides and 0.05 hectare of annual land use. Outputs related to these functional units are approx. 3,000 kg of waste water from coffee washing, 8,500 kg of waste water from the wet method and 750 kg of organic residue that is reincorporated to the tillage as fertilizer. The publication of an LCI of agricultural products is a fundamental step for understanding the potential environmental impacts of each tillage and then establishes the basis for product sustainability. In this way, this work is the first Brazilian initiative for applying LCA to coffee cultivation.

Recommendation and Perspective

Different agricultural practices demonstrate different environmental profiles. The amount of agricultural pesticide is directly related to agricultural practices as tillage rotation, density of plants, etc. This study supplied important results for a better correlation of the agricultural practices and potential environmental impacts of coffee. Future updates of this study will show the evolution of the natural resource management such as land use, new agricultural practices, lower fertilizers and chemicals use.     

Interplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex

Kinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules. Peaking at 15 min, the parasite-induced leakage was drastically enhanced by captopril (Cap), an inhibitor of angiotensin-converting enzyme (ACE), a kinin-degrading metallopeptidase. The enhanced microvascular responses were cancelled by HOE-140, an antagonist of the B2 bradykinin receptor (B2R), or by pre-treatment of promastigotes with the irreversible cysteine proteinase inhibitor N-methylpiperazine-urea-Phe-homoPhe-vinylsulfone-benzene (N-Pip-hF-VSPh). In agreement with the above-mentioned data, the promastigotes vigorously induced edema in the paw of Cap-treated J129 mice, but not Cap-B2R-/- mice. Analysis of parasite-induced breakdown of high molecular weight kininogens (HK), combined with active site-affinity-labeling with biotin-N-Pip-hF-VSPh, identified 35-40 kDa proteins as kinin-releasing cysteine peptidases. We then checked if macrophage infectivity was influenced by interplay between these kinin-releasing parasite proteases, kininogens, and kinin-degrading peptidases (i.e. ACE). Our studies revealed that full-fledged B2R engagement resulted in vigorous increase of L. chagasi uptake by resident macrophages. Evidence that inflammatory macrophages treated with HOE-140 became highly susceptible to amastigote outgrowth, assessed 72 h after initial macrophage interaction, further suggests that the kinin/B2R activation pathway may critically modulate inflammation and innate immunity in visceral leishmaniasis.     

Resistance of three co-occurring resprouter Erica species to highly frequent disturbance

The resistance to experimental, highly frequent disturbance has been analysed in three congeneric, strong-resprouter species (Erica australis, E. scoparia and E. arborea) that co-occur in heath-dominated communities of the northern side of the Strait of Gibraltar, southern Spain. To do so, mature individuals of the three species from a long undisturbed location were clipped at the ground level every sixth month during two years. The relationship between the resprouted biomass dry weight (as indicative of the resprouting vigour) and the upper surface area of the lignotuber along the experiment was established separately for each species at each clipping event by means of linear regressions analysis. The resprouting vigour of the three species was compared by means of independent one-way ANOVAs within each clipping event. Resprouting vigour decreased after recurrent clippings in the three species. Nevertheless, significant differences between species in this loss of resprouting vigour were detected, being E. scoparia the most resistant to the experimental, highly frequent clipping. It is concluded that experimental levels of recurrent disturbance may help to find out differences in resilience within similar (taxonomically, morfologically and/or ecologically), strong-resprouter plant species. Considering the history of forestry management in the nothern side of the Strait of Gibraltar, differences in this regard between the three Erica species may contribute to explain their somewhat segregated ecological distribution in this region.