The molecular analysis of Trypanosoma cruzi metallocarboxypeptidase 1 provides insight into fold and substrate specificity

Trypanosoma cruzi is the aetiological agent of Chagas' disease, a chronic infection that affects millions in Central and South America. Proteolytic enzymes are involved in the development and progression of this disease and two metallocarboxypeptidases, isolated from T. cruzi CL Brener clone, have recently been characterized: TcMCP-1 and TcMCP-2. Although both are cytosolic and closely related in sequence, they display different temporary expression patterns and substrate preferences. TcMCP-1 removes basic C-terminal residues, whereas TcMCP-2 prefers hydrophobic/aromatic residues. Here we report the three-dimensional structure of TcMCP-1. It resembles an elongated cowry, with a long, deep, narrow active-site cleft mimicking the aperture. It has an N-terminal dimerization subdomain, involved in a homodimeric catalytically active quaternary structure arrangement, and a proteolytic subdomain partitioned by the cleft into an upper and a lower moiety. The cleft accommodates a catalytic metal ion, most likely a cobalt, which is co-ordinated by residues included in a characteristic zinc-binding sequence, HEXXH and a downstream glutamate. The structure of TcMCP-1 shows strong topological similarity with archaeal, bacterial and mammalian metallopeptidases including angiotensin-converting enzyme, neurolysin and thimet oligopeptidase. A crucial residue for shaping the S(1') pocket in TcMCP-1, Met-304, was mutated to the respective residue in TcMCP-2, an arginine, leading to a TcMCP-1 variant with TcMCP-2 specificity. The present studies pave the way for a better understanding of a potential target in Chagas' disease at the molecular level and provide a template for the design of novel therapeutic approaches.     

Rapid TLC/GC-MS identification of acetylcholinesterase inhibitors in alkaloid extracts

Alkaloid extracts from 12 plant species of the families Amaryllidaceae, Fumariacae and Papaveraceae were studied with respect to their acetylcholinesterase inhibitory activity and alkaloid patterns. Fifty-three alkaloids were identified by GC-MS, including known acetylcholinesterase (AChE) inhibitors such as galanthamine, epigalanthamine, sanguinine and epinorgalanthamine in extracts of Amaryllidaceae plants and protopine in extracts of Fumariaceae and Papaveraceae plants. The galanthamine-containing extracts of the amaryllidaceous plants were found to be the most active while the extract of Corydalis bulbosa was the most active among the extracts of the tested plants from the Fumariaceae and Papaveraceae plants. TLC bioautographic assay, preparative TLC and GC-MS analysis were combined to identify the active compounds in the studied extracts. Galanthamine was isolated from the known AChE inhibitors in the extracts of Amaryllidaceae plants. Corydaline, bulbocapnine and stylopine were found to be active in the extracts of plant species of the families Fumariaceae and Papaveraceae. Available standards of deshydrocorydaline--a precursor of corydaline, corydaline and stylopine--were tested for AChE inhibitory activity. Deshydrocorydaline and corydaline showed potent inhibitory activity comparable with that of the positive control galanthamine.     

Tumor destruction using electrochemotherapy followed by CpG oligodeoxynucleotide injection induces distant tumor responses

PURPOSE: Electrochemotherapy (ECT) is an effective local therapy of human cutaneous cancers but has no effect on distant untreated tumors. We addressed whether tumor-associated antigens released after ECT could induce an efficient systemic immunity when associated with an appropriate immunoadjuvant. METHODS AND RESULTS: We first studied the nature of the cellular recruitment and the expression of various toll-like receptors (TLRs) in tumors treated by ECT. We found that ECT induced a massive recruitment of CD11c and CD11b positive cells in the tumors and a strong increase of TLR9 expression. We then tested antitumor effects of the combination: ECT followed by TLR-9 ligands, CpG oligodeoxynucleotides (CpG ODN), in three murine tumor models. We found that this combination triggered both potent local synergistic antitumor effects, on the ipsi-lateral ECT-treated tumor, and more interestingly, a systemic antitumor response on the contra-lateral untreated tumor, in the three models. The systemic protection was T-cell dependent as it was not observed in nude littermates. The combination induced tumor-specific T cell effectors in the tumor-draining lymph nodes and in the spleen which secreted significantly more gamma-interferon upon activation than with ECT or CpG ODN alone. CONCLUSIONS: Our data show that ECT and CpG ODN synergize and induce a significant increase of the local effect and a systemic T-dependent antitumor response. Such combination constitutes a potential innovative vaccination strategy using in situ tumor-associated antigens that could eventually be translated into the clinic.     

The Genetic Legacy of Religious Diversity and Intolerance: Paternal Lineages of Christians, Jews, and Muslims in the Iberian Peninsula

Most studies of European genetic diversity have focused on large-scale variation and interpretations based on events in prehistory, but migrations and invasions in historical times could also have had profound effects on the genetic landscape. The Iberian Peninsula provides a suitable region for examination of the demographic impact of such recent events, because its complex recent history has involved the long-term residence of two very different populations with distinct geographical origins and their own particular cultural and religious characteristics—North African Muslims and Sephardic Jews. To address this issue, we analyzed Y chromosome haplotypes, which provide the necessary phylogeographic resolution, in 1140 males from the Iberian Peninsula and Balearic Islands. Admixture analysis based on binary and Y-STR haplotypes indicates a high mean proportion of ancestry from North African (10.6%) and Sephardic Jewish (19.8%) sources. Despite alternative possible sources for lineages ascribed a Sephardic Jewish origin, these proportions attest to a high level of religious conversion (whether voluntary or enforced), driven by historical episodes of social and religious intolerance, that ultimately led to the integration of descendants. In agreement with the historical record, analysis of haplotype sharing and diversity within specific haplogroups suggests that the Sephardic Jewish component is the more ancient. The geographical distribution of North African ancestry in the peninsula does not reflect the initial colonization and subsequent withdrawal and is likely to result from later enforced population movement—more marked in some regions than in others—plus the effects of genetic drift.     

Geographical and technological differences in life cycle inventories shown by the use of process models for waste incinerators part I. technological and geographical differences

Goal and Background  Geographical and technological differences in Life Cycle Inventory data are an important source for uncertainty in the result of Life Cycle Assessments. Knowledge on their impact on the result of an LCA is scarce, and also knowledge on how to manage them in an LCA case study. Objective  Goal of this paper is to explore these differences for municipal solid waste incinerator plants, and to develop recommendations for managing technological and geographical differences. Methodology  The paper provides a definition of technological and geographical differences, and analyses their possible impacts. In a case study, the differences are caused intentionally in ‘games’, by virtually transplanting incineration plants to a different location and by changing parameters such as the composition of the waste input incinerated. The games are performed by using a modular model for municipal solid waste incinerator plants. In each case, an LCA including an Impact Assessment is calculated to trace the impact of these changes, and the results are compared. Conclusions  The conclusions of the paper are two-fold: (1) reduce the differences in inventory data where their impact on the result is high; where it is possible reducing them to a great extent, and the effort for performing the change acceptable; in the case of incineration plants: Adapt the flue gas treatment, especially a possible DeNOx step, to the real conditions; (2) make use of modular process models that allow adapting plant parameters to better meet real conditions, but be aware of possible modelling errors. The paper invites the scientific community to validate the model used for a waste incinerator plant, and suggest putting up similar models for other processes, preferably those of similar relevance for Life Cycle Inventories.     

Elucidation of a monovalent cation dependence and characterization of the divalent cation binding site of the fosfomycin resistance protein (FosA).

The fosfomycin resistance protein FosA is a member of a distinct superfamily of metalloenzymes containing glyoxalase I, extradiol dioxygenases, and methylmalonyl-CoA epimerase. The dimeric enzyme, with the aid of a single mononuclear Mn2+ site in each subunit, catalyzes the addition of glutathione (GSH) to the oxirane ring of the antibiotic, rendering it inactive. Sequence alignments suggest that the metal binding site of FosA is composed of three residues: H7, H67, and E113. The single mutants H7A, H67A, and E113A as well as the more conservative mutants H7Q, H67Q, and E113Q exhibit marked decreases in the ability to bind Mn2+ and, in most instances, decreases in catalytic efficiency and the ability to confer resistance to the antibiotic. The enzyme also requires the monovalent cation K+ for optimal activity. The K+ ion activates the enzyme 100-fold with an activation constant of 6 mM, well below the physiologic concentration of K+ in E. coli. K+ can be replaced by other monovalent cations of similar ionic radii. Several lines of evidence suggest that the K+ ion interacts directly with the active site. Interaction of the enzyme with K+ is found to be dependent on the presence of the substrate fosfomycin. Moreover, the E113Q mutant exhibits a kcat which is 40% that of wild-type in the absence of K+. This mutant is not activated by monovalent cations. The behavior of the E113Q mutant is consistent with the proposition that the K+ ion helps balance the charge at the metal center, further lowering the activation barrier for addition of the anionic nucleophile. The fully activated, native enzyme provides a rate acceleration of >10(15) with respect to the spontaneous addition of GSH to the oxirane.