On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca(2+) dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 mu M focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the VIP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since 117 give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that VIZ bury in the inner mitochondrial membrane and the chemically generated 117 cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MET induction and may have implications for the cell death induced by PTZ. (C) 2010 Elsevier Inc. All rights reserved.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosonne-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 Cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Tryponosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Relações filogenéticas das famílias Coreidae e Pentatomidae (Heteroptera: Pentatomomorpha) baseadas nas sequências de genes mitocondriais (Cyt b, COI e 16S), nucleares (28S e 18S) e informações morfológicas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

AtPIN: Arabidopsis thaliana Protein Interaction Network

Background: Protein-protein interactions (PPIs) constitute one of the most crucial conditions to sustain life in living organisms. To study PPI in Arabidopsis thaliana we have developed AtPIN, a database and web interface for searching and building interaction networks based on publicly available protein-protein interaction datasets. Description: All interactions were divided into experimentally demonstrated or predicted. The PPIs in the AtPIN database present a cellular compartment classification (C(3)) which divides the PPI into 4 classes according to its interaction evidence and subcellular localization. It has been shown in the literature that a pair of genuine interacting proteins are generally expected to have a common cellular role and proteins that have common interaction partners have a high chance of sharing a common function. In AtPIN, due to its integrative profile, the reliability index for a reported PPI can be postulated in terms of the proportion of interaction partners that two proteins have in common. For this, we implement the Functional Similarity Weight (FSW) calculation for all first level interactions present in AtPIN database. In order to identify target proteins of cytosolic glutamyl-tRNA synthetase (Cyt-gluRS) (AT5G26710) we combined two approaches, AtPIN search and yeast two-hybrid screening. Interestingly, the proteins glutamine synthetase (AT5G35630), a disease resistance protein (AT3G50950) and a zinc finger protein (AT5G24930), which has been predicted as target proteins for Cyt-gluRS by AtPIN, were also detected in the experimental screening. Conclusions: AtPIN is a friendly and easy-to-use tool that aggregates information on Arabidopsis thaliana PPIs, ontology, and sub-cellular localization, and might be a useful and reliable strategy to map protein-protein interactions in Arabidopsis. AtPIN can be accessed at http://bioinfo.esalq.usp.br/atpin.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

Background: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion. Methods: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like. Results: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V-repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP. Conclusions: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

The plasma membrane calcium pump, its role and regulation: New complexities and possibilities

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2+-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2+-ATPase controls resting levels of [Ca2+](CYT) has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2+-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors. (C) 1999 Elsevier Science Inc.

New mitochondrial and nuclear evidences support recent demographic expansion and an atypical phylogeographic pattern in the Spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae)

Copyright: © 2014 Rodrigues et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Optimization of immunocytochemistry in cytology: comparison of two protocols for fixation and preservation on cytospin and smear preparations

Objective: A new protocol for fixation and slide preservation was evaluated in order to improve the quality of immunocytochemical reactions on cytology slides. Methods: The quality of immunoreactions was evaluated retrospectively on 186 cytology slides (130 direct smears, 56 cytospins) prepared from different cytology samples. Ninety-three of the slides were air dried, stored at -20 °C and fixed in acetone for 10 minutes (Protocol 1), whereas the other 93 were immediately fixed in methanol at -20 °C for at least 30 minutes, subsequently protected with polyethylene glycol (PEG) and stored at room temperature (Protocol 2). Immunocytochemical staining, with eight primary antibodies, was performed on a Ventana BenchMark Ultra instrument using an UltraView Universal DAB Detection Kit. The following parameters were evaluated for each immunoreaction: morphology preservation, intensity of specific staining, background and counterstain. The slides were blinded and independently scored by four observers with marks from 0 to 20. Results: The quality of immunoreactions was better on methanol-fixed slides protected with PEG than on air-dried slides stored in the freezer: X¯ = 14.44 ± 3.58 versus X¯ = 11.02 ± 3.86, respectively (P < 0.001). Conclusion: Immediate fixation of cytology slides in cold methanol with subsequent application of PEG is an easy and straightforward procedure that improves the quality of immunocytochemical reactions and allows the storage of the slides at room temperature.

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

IntroductionLeishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species.MethodsAll rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification.ResultsOf 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmaniainfections (9/55) were found. In addition, eight Meriones libycus and two Tatera indicawere sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculuswere sampled with no infection.ConclusionsThe results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major(GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

Phylogeographical footprints of the Strait of Gibraltar and Quaternary climatic fluctuations in the western Mediterranean: a case study with the greater white-toothed shrew, Crocidura russula (Mammalia: Soricidae).

We used mitochondrial cyt b sequences to investigate the phylogenetic relationships of Crocidura russula (sensu lato) populations across the Strait of Gibraltar, western Europe, Maghreb, and the Mediterranean and Atlantic islands. This revealed very low genetic divergence between European and Moroccan populations. The application of a molecular clock previously calibrated for shrews suggested that the separation of European from Moroccan lineages occurred less than 60 000 bp, which is at least 5 million years (Myr) after the reopening of the Strait of Gibraltar. This means that an overwater dispersal event was responsible for the observed phylogeographical structure. In contrast, genetic analyses revealed that Moroccan populations were highly distinct from Tunisian ones. According to the molecular clock, these populations separated about 2.2 million years ago (Ma), a time marked by sharp alternations of dry and humid climates in the Maghreb. The populations of the Mediterranean islands Ibiza, Pantelleria, and Sardinia were founded from Tunisian populations by overwater dispersal. In conclusion, overwater dispersal across the Strait of Gibraltar, probably assisted by humans, is possible for small terrestrial vertebrates. Moreover, as in Europe, Quaternary climatic fluctuations had a major effect on the phylogeographical structure of the Maghreb biota.

Multiple refugia and barriers explain the phylogeography of the Valais shrew, Sorex antinorii (Mammalia: Soricomorpha), Sorex antinorii (Mammalia: Soricomorpha)

The aim of the present study was to investigate the genetic structure of the Valais shrew (Sorex antinorii) by a combined phylogeographical and landscape genetic approach, and thereby to infer the locations of glacial refugia and establish the influence of geographical barriers. We sequenced part of the mitochondrial cytochrome b (cyt b) gene of 179 individuals of S. antinorii sampled across the entire species' range. Six specimens attributed to S. arunchi were included in the analysis. The phylogeographical pattern was assessed by Bayesian molecular phylogenetic reconstruction, population genetic analyses, and a species distribution modelling (SDM)-based hindcasting approach. We also used landscape genetics (including isolation-by-resistance) to infer the determinants of current intra-specific genetic structure. The phylogeographical analysis revealed shallow divergence among haplotypes and no clear substructure within S. antinorii. The starlike structure of the median-joining network is consistent with population expansion from a single refugium, probably located in the Apennines. Long branches observed on the same network also suggest that another refugium may have existed in the north-eastern part of Italy. This result is consistent with SDM, which also suggests several habitable areas for S. antinorii in the Italian peninsula during the LGM. Therefore S. antinorii appears to have occupied disconnected glacial refugia in the Italian peninsula, supporting previous data for other species showing multiple refugia within southern refugial areas. By coupling genetic analyses and SDM, we were able to infer how past climatic suitability contributed to genetic divergence of populations. The genetic differentiation shown in the present study does not support the specific status of S. arunchi.

False phylogenies on wood mice due to cryptic cytochrome-b pseudogene.

The phylogeny and phylogeography of the Old World wood mice (subgenus Sylvaemus, genus Apodemus, Muridae) are well-documented. Nevertheless, the distributions of species, such as A. fulvipectus and A. ponticus remain dubious, as well as their phylogenetic relationships with A. sylvaticus. We analysed samples of Apodemus spp. across Europe using the mitochondrial cytochrome-b gene (cyt-b) and compared the DNA and amino-acid compositions of previously published sequences. The main result stemming from this study is the presence of a well-differentiated lineage of Sylvaemus including samples of various species (A. sylvaticus, A. fulvipectus, A. ponticus) from distant locations, which were revealed to be nuclear copies of the mitochondrial cyt-b. The presence of this cryptic pseudogene in published sequences is supported by different pathways. This has led to important errors in previous molecular trees and hence to partial misinterpretations in the phylogeny of Apodemus.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.