RESUMO
The Russian Federation is the largest and one of the most ethnically diverse countries in the world, however no centralized reference database of genetic variation exists to date. Such data are crucial for medical genetics and essential for studying population history. The Genome Russia Project aims at filling this gap by performing whole genome sequencing and analysis of peoples of the Russian Federation. Here we report the characterization of genome-wide variation of 264 healthy adults, including 60 newly sequenced samples. People of Russia carry known and novel genetic variants of adaptive, clinical and functional consequence that in many cases show allele frequency divergence from neighboring populations. Population genetics analyses revealed six phylogeographic partitions among indigenous ethnicities corresponding to their geographic locales. This study presents a characterization of population-specific genomic variation in Russia with results important for medical genetics and for understanding the dynamic population history of the world's largest country.
Assuntos
Variação Genética , Adulto , Doenças Transmissíveis/genética , Demografia , Haplótipos , Humanos , Mutação INDEL , Farmacogenética , Fenótipo , Filogeografia , Polimorfismo de Nucleotídeo Único , Federação Russa/etnologia , Seleção Genética , Sequenciamento Completo do GenomaRESUMO
This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA-CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.
Assuntos
Quitina/análogos & derivados , Pisum sativum/metabolismo , Proteínas de Plantas/metabolismo , Animais , Linhagem Celular , Parede Celular/metabolismo , Parede Celular/microbiologia , Quitina/metabolismo , Quitosana , Hidrólise , Insetos/metabolismo , Micorrizas/metabolismo , Oligossacarídeos , Pisum sativum/microbiologia , Imunidade Vegetal/fisiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Células Sf9 , Transdução de Sinais/fisiologia , Simbiose/fisiologiaRESUMO
Since chitooligosaccharides (COs) are water-soluble, biodegradable and nontoxic compounds, their application may be considered as a promising plant-protecting agent. However, the molecular and cellular modes of action of COs are not yet understood. In this study, transcriptional changes in pea roots treated with COs were investigated via RNA sequencing. Pea roots treated with the deacetylated CO8-DA at low concentration (10-5 Ð) were harvested 24 h after treatment and their expression profiles were compared against medium-treated control plants. We observed 886 differentially expressed genes (fold change ≥ 1; p-value < 0.05) 24 h after treatment with CO8-DA. Gene Ontology term over-representation analysis allowed us to identify the molecular functions of the genes activated in response to CO8-DA treatment and their relation to biological processes. Our findings suggest that calcium signaling regulators and MAPK cascade play a key role in pea plant responses to treatment. Here we found two MAPKKKs, the PsMAPKKK5 and PsMAPKKK20, which might function redundantly in the CO8-DA-activated signaling pathway. In accordance with this suggestion, we showed that PsMAPKKK knockdown decreases resistance to pathogenic Fusarium culmorum fungi. Therefore, analysis showed that typical regulators of intracellular signal transduction pathways involved in triggering of plant responses via CERK1 receptors to chitin/COs in Arabidopsis and rice may also be recruited in legume pea plants.
RESUMO
Genes can be transferred horizontally between prokaryotes and eukaryotes in nature. The best-studied examples occur between Agrobacterium rhizogenes and certain Nicotiana spp. To investigate possible additional cases of horizontal gene transfer in nature between Agrobacterium and plants, a real-time polymerase chain reaction-based approach was employed to screen 127 plant species, belonging to 38 families of Dicotyledones, for the presence of oncogenes homologous to the transfer DNA fragments (T-DNA) from both A. tumefaciens and A. rhizogenes. Among all of the analyzed plant species, we found that only Linaria vulgaris contained sequences homologous to the T-DNA of A. rhizogenes. All screened L. vulgaris plants from various parts of Russia contained the same homologous sequences, including rolB, rolC, ORF13, ORF14, and mis genes. The same opine gene is found in the species of Nicotiana which contain genes of A. rhizogenes. In L. vulgaris, there are two copies of T-DNA organized as a single tandem imperfect direct repeat. The plant DNA sequence of the site of integration shows similarity to a retrotransposon. This site is most likely silent, suggesting that the T-DNA is not expressed. Attempts to demonstrate expression of the T-DNA genes were negative. Our study indicates that the frequency of gene transfer and fixation in the germline from Agrobacterium to plant hosts is rare in the natural environment.
Assuntos
Agrobacterium/genética , Transferência Genética Horizontal , Genes Bacterianos/genética , Linaria/genética , Agrobacterium/isolamento & purificação , Sequência de Bases , DNA Bacteriano/genética , DNA de Plantas/genética , Linaria/microbiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Regeneração , Federação Russa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Microsporidia are a group of fungus-related intracellular parasites with severely reduced metabolic machinery. They lack canonical mitochondria, a Krebs cycle, and a respiratory chain but possess genes encoding glycolysis enzymes, a glycerol phosphate shuttle, and ATP/ADP carriers to import host ATP. The recent finding of alternative oxidase genes in two clades suggests that microsporidial mitosomes may retain an alternative respiratory pathway. We expressed the fragments of mitochondrial chaperone Hsp70 (mitHsp70), mitochondrial glycerol-3-phosphate dehydrogenase (mitG3PDH), and alternative oxidase (AOX) from the microsporidium Antonospora (Paranosema) locustae in Escherichia coli. Immunoblotting with antibodies against recombinant polypeptides demonstrated specific accumulation of both metabolic enzymes in A. locustae spores. At the same time comparable amounts of mitochondrial Hsp70 were found in spores and in stages of intracellular development as well. Immunoelectron microscopy of ultrathin cryosections of spores confirmed mitosomal localization of the studied proteins. Small amounts of enzymes of an alternative respiratory chain in merogonial and early sporogonial stages, alongside their accumulation in mature spores, suggest conspicuous changes in components and functions of mitosomes during the life cycle of microsporidia and the important role of these organelles in parasite energy metabolism, at least at the final stages of sporogenesis.
Assuntos
Transporte de Elétrons/fisiologia , Metabolismo Energético , Proteínas Fúngicas/metabolismo , Microsporídios/metabolismo , Organelas/metabolismo , Esporos Fúngicos/metabolismo , Imuno-Histoquímica , Esporos Fúngicos/químicaRESUMO
Heterotrimeric G-proteins regulate plant growth and development as master regulators of signaling pathways. In legumes with indeterminate nodules (e.g., Medicago truncatula and Pisum sativum), the role of heterotrimeric G-proteins in symbiosis development has not been investigated extensively. Here, the involvement of heterotrimeric G-proteins in M. truncatula and P. sativum nodulation was evaluated. A genome-based search for G-protein subunit-coding genes revealed that M. truncatula and P. sativum harbored only one gene each for encoding the canonical heterotrimeric G-protein beta subunits, MtG beta 1 and PsG beta 1, respectively. RNAi-based suppression of MtGbeta1 and PsGbeta1 significantly decreased the number of nodules formed, suggesting the involvement of G-protein beta subunits in symbiosis in both legumes. Analysis of composite M. truncatula plants carrying the pMtGbeta1:GUS construct showed ß-glucuronidase (GUS) staining in developing nodule primordia and young nodules, consistent with data on the role of G-proteins in controlling organ development and cell proliferation. In mature nodules, GUS staining was the most intense in the meristem and invasion zone (II), while it was less prominent in the apical part of the nitrogen-fixing zone (III). Thus, MtG beta 1 may be involved in the maintenance of meristem development and regulation of the infection process during symbiosis. Protein-protein interaction studies using co-immunoprecipitation revealed the possible composition of G-protein complexes and interaction of G-protein subunits with phospholipase C (PLC), suggesting a cross-talk between G-protein- and PLC-mediated signaling pathways in these legumes. Our findings provide direct evidence regarding the role of MtG beta 1 and PsG beta 1 in symbiosis development regulation.
RESUMO
The IPD3/CYCLOPS transcription factor was shown to be involved in the regulation of nodule primordia development and subsequent stages of nodule differentiation. In contrast to early stages, the stages related to nodule differentiation remain less studied. Recently, we have shown that the accumulation of cytokinin at later stages may significantly impact nodule development. This conclusion was based on a comparative analysis of cytokinin localization between pea wild type and ipd3/cyclops mutants. However, the role of cytokinin at these later stages of nodulation is still far from understood. To determine a set of genes involved in the regulation of later stages of nodule development connected with infection progress, intracellular accommodation, as well as plant tissue and bacteroid differentiation, the RNA-seq analysis of pea mutant SGEFix--2 (sym33) nodules impaired in these processes compared to wild type SGE nodules was performed. To verify cytokinin's influence on late nodule development stages, the comparative RNA-seq analysis of SGEFix--2 (sym33) mutant plants treated with cytokinin was also conducted. Findings suggest a significant role of cytokinin in the regulation of later stages of nodule development.
RESUMO
The life cycle, ultrastructure, and molecular phylogeny of a new microsporidium Crispospora chironomi g.n. sp.n., a parasite of the midge Chironomus plumosus, are described. The parasite infects the gut epithelium of the host larvae and possesses sporogonies of two types, polysporoblastic and disporoblastic, respectively, proceeding within the same host cell. In the sporogonial sequence of the first type, dozens of spherical monokaryotic spores within a thick-walled capsule are formed. The spores are 1.5-2.0 µm in diameter; the exospore possesses two to three bundles of tubular protrusions. In the sporogonial sequence of the second type, diplokaryotic oval spores, 2.5 × 1.5 µm in size, are formed within a compartment, partially surrounded with multilayered membranes. Spores of both types are similar in respect to inner structure, possessing a well-developed extrusion apparatus with (a) the anterior vesicular part of the polaroplast covering the lamellar posterior one and (b) isofilar polar filament with several coils in one row. Small subunit ribosomal DNA phylogeny showed position of the new microsporidium in a cluster uniting microsporidia of terrestrial origin infecting diverse hosts, nested within Clade IV, corresponding to Class Terresporidia sensu Vossbrinck and Debrunner-Vossbrinck (Folia Parasitol 52:131-142, 2005).
Assuntos
Chironomidae/microbiologia , Microsporídios não Classificados/classificação , Microsporídios não Classificados/ultraestrutura , Animais , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Mucosa Intestinal/microbiologia , Estágios do Ciclo de Vida , Microsporídios não Classificados/genética , Microsporídios não Classificados/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNA , Esporos Fúngicos/ultraestruturaRESUMO
Larvae of Chironomus plumosus, collected in North-Western Russia in September 2008, were infected with a microsporidium possessing broadly oval uninucleate spores in sporophorous vesicles. Sporogony and spore ultrastructure of this microsporidium differed from that of known microsporidian species, suggesting establishment of a new species, Anisofilariata chironomi, being a type species of a new genus. Sporogony di-, tetra-, octo-, and 16-sporoblastic. Fixed and stained spores are 4.7-6.8 x 3.4-5.4 microm in size, the spore measurements varying depending upon the number of spores in the sporophorous vesicle. The polaroplast is bipartite, with anterior and posterior parts composed of very thin and thick lamellae, respectively, and occupies the major volume of the spore. The polar filament is anisofilar, with two broad proximal and 10-13 narrow distal coils arranged in 2-4 layers. The sporophorous vesicle is bounded by a thin membrane and contains multiple tubular structures. Small subunit ribosomal DNA phylogeny showed basal position of the new microsporidium to a cluster uniting microsporidia infecting ciliates (Euplotespora binucleata), microcrustaceans (Glugoides intestinalis, Mrazekia macrocyclopis), lepidopteran insects (Cystosporogenes spp., Endoreticulatus spp.) and human (Vittaforma corneae), nested within Clade IV sensu Vossbrinck and Debrunner-Vossbrinck (2005 Folia Parasitol 52:131-142). No close phylogenetic relationships were found between A. chironomi and microsporidia from other dipteran hosts.
Assuntos
Chironomidae/microbiologia , Microsporídios não Classificados/classificação , Microsporídios não Classificados/ultraestrutura , Filogenia , Animais , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Microscopia , Microscopia Eletrônica de Transmissão , Microsporídios não Classificados/genética , Microsporídios não Classificados/isolamento & purificação , Dados de Sequência Molecular , Organelas/ultraestrutura , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Federação Russa , Análise de Sequência de DNA , Esporos Fúngicos/ultraestruturaRESUMO
A novel radioligand, 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K(d) and T(1/2) of dissociation of NIDA522131 binding measured at 37 degrees C in vitro were 4.9 +/- 0.4 pmol/L and 81 +/- 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the alpha(4)beta(2)* nAChRs distribution. Comparison between [(18)F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [(18)F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3-4 fold higher for [(18)F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at alpha(4)beta(2)* versus alpha(3)beta(4)* nAChRs. These results suggest that [(18)F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.
Assuntos
Azetidinas/farmacologia , Radioisótopos de Flúor/farmacocinética , Piridinas/farmacologia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Tálamo/diagnóstico por imagem , Animais , Autorradiografia , Azetidinas/química , Comportamento Animal/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estudos de Avaliação como Assunto , Radioisótopos de Flúor/química , Técnicas In Vitro , Macaca mulatta , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Plasma/efeitos dos fármacos , Tomografia por Emissão de Pósitrons/métodos , Piridinas/química , Compostos Radiofarmacêuticos/química , RatosRESUMO
Quantification of alpha(4)beta(2)* nicotinic acetylcholine receptors using 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]FA) and positron emission tomography (PET) imaging requires measurement of nonmetabolized radioligand in blood plasma, which was previously accomplished using high-performance liquid chromatography (HPLC). Here, we introduce a one-step solid-phase extraction (SPE) method for measuring the concentration of nonmetabolized 2-[(18)F]FA. This method allows many samples to be processed in a short period of time. SPE effectively separated 2-[(18)F]FA from radioactive metabolites typically observed in blood plasma after administration of radioligand in humans. Measurements of the 2-[(18)F]FA parent fraction in healthy human volunteers obtained using the SPE method were nearly identical to those obtained using HPLC (1.3+/-5% average underestimation of SPE), and reproducibility was good within and between runs (2% and 6% coefficient of variation, respectively). SPE recovery of 2-[(18)F]FA from blood plasma was not appreciably diminished (3+/-0.6%) by a larger volume of blood plasma loaded onto the cartridge, suggesting the possibility of increasing the plasma sample volume at later times in a PET study to improve measurement sensitivity. 2-[(18)F]FA was stable in blood stored on ice over 8 h and in saline at low concentrations (<2 MBq/ml) but not at high concentrations (ca. 130 MBq/ml). Using SPE, the elimination half-life and full body distribution volume of 2-[(18)F]FA in healthy human volunteers were estimated as 4.2+/-0.8 h and 220+/-70 L, respectively. These results suggest that SPE is the method of choice for the determination of the plasma 2-[(18)F]FA concentration when measurement of individual metabolites is not required.
Assuntos
Azetidinas/sangue , Análise Química do Sangue/métodos , Piridinas/sangue , Extração em Fase Sólida/métodos , Animais , Haplorrinos , Humanos , Compostos Radiofarmacêuticos/sangue , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A new series of CB(1) ligands with high binding affinity (K(i) = 0.7-100 nM) and moderate lipophilicity (cLogD(7.4)) in the range of 2.1-4.5 has been synthesized. A structure-activity relationship study demonstrated that for the studied set of aminoalkylindoles, the molecular dipole of the ground state conformation within the series was inversely related to the affinity. The racemic ligand with highest affinity (0.7 nM), 3-(4-fluoronaphthoyl)-1-(N-methylpiperidin-2-ylmethyl)indole, was radiolabeled with (18)F. This radioligand specifically labeled CB(1) receptors in mouse brain and accumulated in regions of high versus low CB(1) receptor density in a ratio of 1.6. The displaceable radioactivity of one enantiomer in the brains of mice determined in a pretreatment study using the CB(1) antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716) was nearly double that of the racemate for the same determination; therefore, the active enantiomer is a candidate for PET studies in animals. A pretreatement study for the other enantiomer found no displaceable radioactivity in the same group of mice; this result suggested the enantiomer was inactive.
Assuntos
Encéfalo/metabolismo , Indóis/síntese química , Neurônios/metabolismo , Piperidinas/síntese química , Compostos Radiofarmacêuticos/síntese química , Receptor CB1 de Canabinoide/metabolismo , Animais , Cerebelo/metabolismo , Radioisótopos de Flúor , Técnicas In Vitro , Indóis/química , Indóis/farmacocinética , Ligantes , Masculino , Camundongos , Modelos Moleculares , Piperidinas/química , Piperidinas/farmacocinética , Tomografia por Emissão de Pósitrons , Ensaio Radioligante , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Reportedly, 2-[(18)F]fluoro-A-85380, 1, a promising radiotracer for imaging the nicotinic acetylcholine receptor (nAChR) by positron emission tomography (PET) in humans, exhibits slow penetration through the blood-brain barrier (BBB) due to its low lipophilicity. A ligand for nAChRs with greater lipophilicity than that of 1 would be potentially more favorable for PET imaging of nAChR due to its faster penetration through the BBB. Herein, a novel series of compounds has been developed based on the high affinity ligand for nAChRs, 2-chloro-5-((1-methyl-2-(S)-pyrrolidinyl)methoxy)-3-(2-(4-pyridinyl)vinyl)pyridine, 3b. The in vitro binding affinities for the new series were found to be in the range of K(i) = 9-331 pM. A molecular modeling study showed differences in the comformational profiles and the electronic properties of these compounds, which provides further insight into the structure-activity relationships at nAChR. Lipophilicities of the compounds 3b-6b have been found to be substantially higher than that of 1. As a result, compounds 3b-6b might exhibit a faster penetration through the BBB than the less lipophilic 1. The N-methyl derivatives 3b and 6b demonstrated very high affinities at nAChRs (K(i) = 28 and 23 pM, respectively) and will be targets for development of (11)CH(3)-labeled derivatives as radiotracers for PET imaging of nAChRs.
Assuntos
Piridinas/síntese química , Pirrolidinas/síntese química , Receptores Nicotínicos/metabolismo , Animais , Ligação Competitiva , Encéfalo/metabolismo , Técnicas In Vitro , Ligantes , Modelos Moleculares , Conformação Molecular , Piridinas/química , Piridinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Ensaio Radioligante , Ratos , Relação Estrutura-Atividade , Tomografia Computadorizada de EmissãoRESUMO
Potential positron emission tomography (PET) ligands with low picomolar affinity at the nicotinic acetylcholine receptor (nAChR) and with lipophilicity (log D) ranging from -1.6 to +1.5 have been synthesized. Most members of the series, which are derivatives of 5-substituted-6-halogeno-A-85380, exhibited a higher binding affinity at alpha4beta2-nAChRs than epibatidine. An analysis, by molecular modeling, revealed an important role of the orientation of the additional heterocyclic ring on the binding affinity of the ligands with nAChRs. The existing nicotinic pharmacophore models do not accommodate this finding. Two compounds of the series, 6-[(18)F]fluoro-5-(pyridin-3-yl)-A-85380 ([(18)F]31) and 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-[(18)F]fluoropyridin-5-yl)pyridine) ([(18)F]35), were radiolabeled with (18)F. Comparison of PET data for [(18)F]31 and 2-[(18)F]FA shows the influence of lipophilicity on the binding potential. Our recent PET studies with [(18)F]35 demonstrated that its binding potential values in Rhesus monkey brain were ca. 2.5 times those of 2-[(18)F]FA. Therefore, [(18)F]35 and several other members of the series, when radiolabeled, will be suitable for quantitative imaging of extrathalamic nAChRs.
Assuntos
Azetidinas/síntese química , Piridinas/síntese química , Pirrolidinas/síntese química , Compostos Radiofarmacêuticos/síntese química , Receptores Nicotínicos/metabolismo , Animais , Azetidinas/química , Azetidinas/farmacologia , Ligação Competitiva , Encéfalo/metabolismo , Radioisótopos de Flúor , Técnicas In Vitro , Marcação por Isótopo , Ligantes , Macaca mulatta , Modelos Moleculares , Piridinas/química , Piridinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Ensaio Radioligante , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacologia , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-Atividade , Tomografia Computadorizada de Emissão/métodosRESUMO
Exploration of the central CB1 cannabinoid receptors using positron emission tomography (PET) will allow for an understanding of the pharmacological and physiological role played by these receptors in the CNS. Current tracers are highly lipophilic compounds that exhibit very high nonspecific to specific binding ratios and as a result are inapt for use in humans. We have synthesized a series of less lipophilic analogues of SR141716 to serve as potential radioligands. Binding affinities of the series and a functional electrophysiological assay of three of our compounds have been presented.
Assuntos
Canabinoides/metabolismo , Piperidinas/síntese química , Pirazóis/síntese química , Receptores de Droga/metabolismo , Animais , Benzoxazinas , Cerebelo/metabolismo , Técnicas In Vitro , Ligantes , Potenciais da Membrana , Morfolinas/farmacologia , Naftalenos/farmacologia , Núcleo Accumbens/fisiologia , Piperidinas/química , Piperidinas/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Ensaio Radioligante , Ratos , Receptores de Canabinoides , Receptores de Droga/antagonistas & inibidores , Rimonabanto , Relação Estrutura-Atividade , Tomografia Computadorizada de EmissãoRESUMO
Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/ß-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/ß-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.
Assuntos
Apansporoblastina/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Locusta migratoria/microbiologia , Microsporidiose/metabolismo , Sequência de Aminoácidos , Animais , Apansporoblastina/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/genética , Locusta migratoria/genética , Locusta migratoria/metabolismo , Redes e Vias Metabólicas/genética , Microsporídios/genética , Microsporídios/metabolismo , Microsporidiose/genética , Dados de Sequência Molecular , Filogenia , Pichia/genética , Pichia/metabolismoRESUMO
Microsporidia, a large group of fungi-related intracellular parasites, are characterized by drastically reduced metabolism. They possess genes encoding glycolysis components, and the glycerol-phosphate shuttle, but lack mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except alpha and beta subunits of E(1) enzyme of pyruvate dehydrogenase (PDH) complex. Here, we have expressed PDH subunits from the microsporidum Paranosema (Antonospora) locustae in Escherichia coli. Western blot analysis with antibodies raised against recombinant proteins has revealed their specific accumulation in mature spores of P. locustae but not in the intracellular development stages. Two subunits were coprecipitated as a single heterooligomeric complex by anti-alpha or anti-beta PDH antibodies. Ultracentrifugation of spore homogenate has shown the presence of PDH in the soluble fraction. Relocalization of the mitochondrial protein in microsporidial spore cytoplasm was confirmed by immunoelectron microscopy of ultrathin cryosections with affinity-purified anti-alpha PDH antibodies. On cryosections, parasite enzyme was found partly associated with the cytoplasmic side of ER and other intraspore membranes, suggesting that electrons might be transferred to any membrane acceptor and finally to oxygen in the parasite cell.
Assuntos
Apansporoblastina/enzimologia , Subunidades Proteicas/biossíntese , Piruvato Desidrogenase (Lipoamida)/biossíntese , Apansporoblastina/química , Apansporoblastina/genética , Apansporoblastina/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Imunoprecipitação , Microscopia Imunoeletrônica , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Ligação Proteica , Subunidades Proteicas/genética , Piruvato Desidrogenase (Lipoamida)/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Esporos Fúngicos/química , Esporos Fúngicos/ultraestruturaRESUMO
Nicotinic acetylcholine receptors are involved in tobacco dependence and several other neuropathologies (e.g., Alzheimer's disease, Parkinson's disease), as well as in attention, learning, and memory. Performing in vivo imaging of these receptors in humans holds great promise for understanding their role in these conditions. Recently, three radiohalogenated analogs of 3-(2(S)-azetidinylmethoxy)pyridine (A- 85380) were used successfully for the in vivo visualization of alpha4beta2* nicotinic receptors in the human brain with PET/SPECT. Herein, we present the results of the in vitro characterization of one of these radioligands, 6-[18F]fluoro-3-(2(S)-azetidinylmethoxy)-pyridine (6-[18F]fluoro-A-85380), which is a fluoro-analog of the potent nonopioid analgesic ABT-594. In human postmortem cortical tissue, 6-[18F]fluoro-A-85380 reversibly binds with high affinity to a single population of sites (Kd = 59 pM at 37 degrees C, Bmax = 0.7 pmol/g tissue). The binding is fully reversible and is characterized at 37 degrees C by T(1/2assoc) = 2.2 min (at a ligand concentration of 39 pM) and by T(1/2dissoc) = 3.6 min. 6-Fluoro-A-85380 exhibits clear selectivity for alpha4beta2* over the other major mammalian nicotinic receptor subtypes: alpha7, alpha3beta4, and muscle-type. These results suggest that 6-[18F]fluoro-A-85380 is a promising radioligand for in vivo imaging of brain alpha4beta2* nicotinic receptors.
Assuntos
Azetidinas/farmacocinética , Encéfalo/metabolismo , Radioisótopos de Flúor/farmacocinética , Ligantes , Receptores Nicotínicos/metabolismo , Animais , Azetidinas/química , Ligação Competitiva/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Órgão Elétrico/metabolismo , Epistasia Genética , Radioisótopos de Flúor/química , Humanos , Técnicas In Vitro , Isótopos de Iodo/farmacocinética , Mudanças Depois da Morte , Ensaio Radioligante/métodos , Cintilografia , Ratos , Temperatura , Fatores de Tempo , Distribuição Tecidual , TorpedoRESUMO
External imaging of nicotinic acetylcholine receptors (nAChRs) using techniques such as PET would help to clarify the roles of these receptors in the physiology and pathology of brain function. Here we report the results of quantitative PET studies of cerebral nAChRs with 2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) in rhesus monkeys. Data from dynamic PET scans were analyzed using graphical methods. Binding potential (BP) values of 2.0, 0.4, 0.3, and 0.03 observed in the thalamus (Th), cortex (Cx), striatum (Str), and cerebellum (Cb), respectively, were consistent with the pattern of alpha(4)beta(2) nAChR distribution in monkey brain. The high value of 2-[(18)F]FA-specific binding in the rhesus monkey Th and low level of that in Cb compared with nonspecific accumulation of radioactivity in these structures allowed use of Cb as a reference region for calculation of BP and volume of distribution of specific binding (VDsb) in Th by graphical methods, both with and without the plasma input function. In contrast, estimation of 2-[(18)F]FA specific binding in low-receptor-density regions such as Cx and Str required assessment of nondisplaceable volume of distribution (VDnd) in a separate study and measurement of nonmetabolized radioligand concentrations in the plasma. For accurate quantitation of 2-[(18)F]FA-specific binding by graphical analysis, PET studies should last up to 7 h due to the slow kinetics of 2-[(18)F]FA brain distribution. Further, to avoid substantial underestimation in measured BP values the doses of administered 2-[(18)F]FA should not exceed 0.1 nmol/kg body weight. The findings suggest that 2-[(18)F]FA is a promising ligand for quantitation of nAChRs in human brain.