Leishmaniasis in central and southern Tunisia: current geographical distribution of zymodemes

The authors report the identification of Leishmania strains isolated from the Centre and the South of Tunisia. 266 strains were isolated between 1998 and 2006 from human (n=221 strains) and dogs (n=45 strains) hosts. The isoenzymatic identification exhibits the presence of in total five zymodemes belonging to three Leishmanio complexes: Leishmania infantum, L. major and L. killicki. All strains isolated from human and canine visceral leishmaniasis belonged to L. infantum. zymodeme MON-1 was the only one isolated from canine visceral leishmaniasis. However, it is predominant in human visceral leishmaniasis beside zymodeme MON-24 which was detected in two provinces of the Centre (Monastir and Kairouan) and zymodeme MON-80 isolated for the first time in Kairouan province. Three complexes are responsible for human cutaneous leishmaniasis: L. major MON-25 is the parasite the most frequently found in its classic foci in the Centre and the South of the country. L. infantum MON-24 was isolated for the first time in a small locality of Sfax (southern Tunisia) showing the appearance of a new focus of L. infantum. L. killicki was isolated in its original focus of Tataouine and in two new foci of the central part of the country (Sidi Bouzid and Kairouan).     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Population structure of Tunisian Leishmania infantum and evidence for the existence of hybrids and gene flow between genetically different populations

Twenty-seven strains of Leishmania infantum from north and central Tunisia belonging to the three main MON zymodemes (the MON-typing system is based on multilocus enzyme electrophoresis (MLEE) of 15 enzymes) found in this country (MON-1, MON-24 and MON-80) and representing different pathologies (visceral, cutaneous and canine leishmaniasis) have been studied to understand the genetic polymorphism within this species. Intraspecific variation could be detected in L. infantum by the use of 14 hypervariable microsatellite markers. In addition to microsatellite repeat length variation, a high degree of allelic heterozygosity has been observed among the strains investigated, suggestive of sexual recombination within L. infantum groups. The two major clusters found by using Bayesian statistics as well as distance analysis are consistent with the classification based on isoenzymes, dividing Tunisian L. infantum into MON-1 and MON-24/MON-80. Moreover, the existence of hybrid strains between the MON-1 and the non-MON-1 populations has been shown and verified by analysis of clones of one of these strains. Substructure analysis discriminated four groups of L. infantum. The major MON-1 cluster split into two groups, one comprising only Tunisian strains and the second both Tunisian and European strains. The major MON-24 cluster was subdivided into two groups with geographical and clinical feature correlations: a dermotropic group of strains mainly from the north, and a viscerotropic group of strains from the centre of Tunisia. The four viscerotropic hybrid strains all originated from central Tunisia and were typed by MLEE as MON-24 or MON-80. To our knowledge, this is the first report describing relationships between clinical picture and population substructure of L. infantum MON-24 based on genotype data, as well as the existence of hybrids between zymodemes MON-1 and MON-24/MON-80, and proving one of these hybrid strains by molecular analysis of the parent strain and its clones.     

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.     

Faunistic note on Culcoides (Diptera, Ceratopogonidae) from the governate of Monastir (Tunisia)

Following the arrival of blue-tongue in Tunisia, the authors report the results of the first survey made in Monastir. They show the existence of nine species of Culicoides, three of which are new to the country IC. paolae, C. imicola, C. newsteadi), that now brings to 22 the number of the known species.     

Antimicrobial mechanism of action of surfactant lipid preparations in enteric Gram-negative bacilli

Two surfactant lipid preparations (SLPs) were investigated to determine their mechanism of antimicrobial action. 8N8, a water-in-oil emulsion, and W60C, a liposome, both have bactericidal activity against Gram-positive bacteria and non-enteric Gram-negative bacteria. Additionally, W60C is bactericidal for enteric Gram-negative bacilli when suspended in deionized water. Zeta potential measurements suggested that the resistance of Gram-negative bacilli to 8N8 might be caused by ionic repulsion. Addition of 50 micromol 1(-1) ethylene diamine tetra acetic acid in 100 mmol 1(-1) Tris buffer to either SLPs yielded efficient bactericidal activity against Gram-negative bacilli. This appeared to be due to disruption of the outer membrane and the chelation of divalent cations, as the addition of excess calcium inhibited the antimicrobial effect. Electron microscopy studies documented that 8N8 disrupts the bacterial cell wall, lysing the bacteria, while W60C fuses and internalizes within the cell, causing damage without immediate cell lysis. Understanding the mechanisms of action of these biocidal formulations will help to produce improved formulations with broader spectra of activity.     

3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.     

Identifying the lipid-protein interface of the alpha4beta2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.