Helminths of roe deer (Capreolus capreolus) in the Middle Black Sea Region of Turkey

Fifteen roe deer were examined at necropsy from Northern Turkey in the period 2006-2010 for the helminth infections. Totally 6470 helminth specimens were collected and identified by morphological criteria. Twenty-five helminth species were identified (1 of the Class Trematoda, 1 of Cestoda and 23 of Nematoda). Dicrocoelium dendriticum (Prevalence 20%) was found in liver. Cysticercus tenuicollis (6.6%) was found in mesentery. Haemonchus contortus (53.3%), Ostertagia leptospicularis (73.3%), O. leptospicularis (minor morph: kolchida) (53.3%), Ostertagia ostertagi (26.6%), Spiculopteragia spiculoptera (66.6%), S. spiculoptera (minor morph: mathevossiani) (6.6%), Teladorsagia circumcincta (40.0%), T. circumcincta (minor morph: davtiani) (6.6%), T. circumcincta (minor morph: trifurcata) (6.6%), Trichostrongylus axei (66.6%) were found in abomasum. Trichostrongylus andreevi (6.6%), T. colubriformis (6.6%), T. longispicularis (26.6%), T. vitrinus (40.0%), T. capricola (6.6%), Cooperia oncophora (26.6%), C. punctata (6.6%), Nematodirus filicollis (66.6%), and Capillaria bovis (26.6%) were found in small intestine. Oesophagostomum venulosum (46.6%), Chabertia ovina (26.6%), and Trichuris ovis (13.3%) were found in large intestine. Dictyocaulus capreolus (6.6%) was found in lungs.     

Detection and characterization of Wolbachia infections in laboratory and natural populations of different species of tsetse flies (genus Glossina)

Background

Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals.

Results

In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome.

Conclusions

Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods.

     

Extending signaling pathways with protein-interaction networks. Application to apoptosis

Cells exploit signaling pathways during responses to environmental changes, and these processes are often modulated during disease. Particularly, relevant human pathologies such as cancer or viral infections require downregulating apoptosis signaling pathways to progress. As a result, the identification of proteins responsible for these changes is essential for the diagnostics and development of therapeutics. Transferring functional annotation within protein interaction networks has proven useful to identify such proteins, although this is not a trivial task. Here, we used different scoring methods to transfer annotation from 53 well-studied members of the human apoptosis pathways (as known by 2005) to their protein interactors. All scoring methods produced significant predictions (compared to a random negative model), but its number was too large to be useful. Thus, we made a final prediction using specific combinations of scoring methods and compared it to the proteins related to apoptosis signaling pathways during the last 5 years. We propose 273 candidate proteins that may be relevant in apoptosis signaling pathways. Although some of them have known functions consistent with their proposed apoptotsis involvement, the majority have not been annotated yet, leaving room for further experimental studies. We provide our predictions at http://sbi.imim.es/web/Apoptosis.php.     

Cooperativity in oxidation reactions catalyzed by cytochrome P450 1A2: highly cooperative pyrene hydroxylation and multiphasic kinetics of ligand binding

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of alpha-naphthoflavone (alphaNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for alphaNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (alphaNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-alphaNF complex show active site space for only one alphaNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.     

Modified glutamine catabolism in macrophages of Ucp2 knock-out mice

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.     

Cooperativity of cytochrome P450 1A2: interactions of 1,4-phenylene diisocyanide and 1-isopropoxy-4-nitrobenzene

Homotropic cooperativity of 1-alkoxy-4-nitrobenzene substrates and also their heterotropic cooperative binding interactions with the iron ligand 1,4-phenylene diisocyanide (Ph(NC)₂) had been demonstrated previously with rabbit cytochrome P450 (P450) 1A2 [G.P. Miller, F.P. Guengerich, Biochemistry 40 (2001) 7262-7272]. Multiphasic kinetics were observed for the binding of Ph(NC)₂ to both ferric and ferrous P450 1A2, including relatively slow steps. Ph(NC)₂ induced an apparently rapid change in the circular dichroism spectrum, consistent with a structural change, but had no effect on tryptophan fluorescence. Ph(NC)₂ binds the P450 iron in both the ferric and ferrous forms; ferric P450 1A2 was reduced rapidly in the absence of added ligands, and the rate was attenuated when Ph(NC)₂ was bound. No oxidation products of Ph(NC)₂ were detected. Docking studies with a rabbit P450 1A2 homology model based on the published structure of a human P450 1A2·α-naphthoflavone (αNF) complex indicated adequate room for a complex with either two 1-isopropoxy-4-nitrobenzene molecules or a combination of one 1-isopropoxy-4-nitrobenzene and one Ph(NC)₂; in the case of αNF no space for an extra ligand was available. The patterns of homotropic cooperativity seen with 1-alkoxy-4-nitrobenzenes (biphasic plots of v vs. S) differ from those seen with polycyclic hydrocarbons (positive cooperativity), suggesting that only with the latter does the ligand interaction produce improved catalysis. Consistent with this view, Ph(NC)₂ inhibited the oxidation of 1-isopropoxy-4-nitrobenzene and other substrates.     

The implementation of SOMO (SOlution MOdeller) in the UltraScan analytical ultracentrifugation data analysis suite: enhanced capabilities allow the reliable hydrodynamic modeling of virtually any kind of biomacromolecule

The interpretation of solution hydrodynamic data in terms of macromolecular structural parameters is not a straightforward task. Over the years, several approaches have been developed to cope with this problem, the most widely used being bead modeling in various flavors. We report here the implementation of the SOMO (SOlution MOdeller; Rai et al. in Structure 13:723–734, 2005) bead modeling suite within one of the most widely used analytical ultracentrifugation data analysis software packages, UltraScan (Demeler in Modern analytical ultracentrifugation: techniques and methods, Royal Society of Chemistry, UK, 2005). The US-SOMO version is now under complete graphical interface control, and has been freed from several constraints present in the original implementation. In the direct beads-per-atoms method, virtually any kind of residue as defined in the Protein Data Bank (e.g., proteins, nucleic acids, carbohydrates, prosthetic groups, detergents, etc.) can be now represented with beads whose number, size and position are all defined in user-editable tables. For large structures, a cubic grid method based on the original AtoB program (Byron in Biophys J 72:408–415, 1997) can be applied either directly on the atomic structure, or on a previously generated bead model. The hydrodynamic parameters are then computed in the rigid-body approximation. An extensive set of tests was conducted to further validate the method, and the results are presented here. Owing to its accuracy, speed, and versatility, US-SOMO should allow to fully take advantage of the potential of solution hydrodynamics as a complement to higher resolution techniques in biomacromolecular modeling.     

A short synthesis of Dronedarone

A modification of the Nenitzescu reaction was used to obtain Dronedarone from quinonimine 20 and 1,3-diketone 14 (R?=?CH₂CH₂CH₂NBu₂) in a two-stage process in almost 55% overall yield. Our results represent significant improvement over other state-of-the-art methods as no extra steps for the decoration of the benzofuran core are required.