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1.
PLoS Negl Trop Dis ; 14(5): e0008242, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32401754

RESUMO

Alveolar and cystic echinococcosis (AE, CE) caused by E. multilocularis and E. granulosus s.l., respectively, are considered emerging zoonotic diseases in Kyrgyzstan with some of the world highest regional incidences. Little is known regarding the molecular variability of both species in Kyrgyzstan. In this study we provide molecular data from a total of 72 parasite isolates derived from humans (52 AE and 20 CE patients) and 43 samples from dogs (23 infected with E. multilocularis and 20 with E. granulosus s.l.).Genetic variability in E. multilocularis was studied using the concatenated complete sequences of the cob, nad2 and cox1 mitochondrial genes adding a total of 3,558bp per isolate. The cob/nad2/cox1 A2 haplotype was identified in 63.4% of the human and in 65.2% of the dog samples. This haplotype was originally described in samples from Kazakhstan and St. Lawrence Island (Alaska, USA). We also describe here 16 non-previously defined variants of E. multilocularis (called A11-A26). All haplotypes cluster together within the Asian group in the haplotype network. Based on Fst values, low level of genetic differentiation was found between the populations of E. multilocularis isolated from different regions within the country. However, high degree of differentiation was found when all the concatenated sequences from Kyrgyzstan are considered as a single population and compared with the population of the parasite from the neighbouring country China. In the case of E. granulosus s.l. the analysis was based in 1,609bp of the cox1 gene. One isolate from a dog was identified as E. equinus, while all the other sequences were identified belonging to E. granulosus s.s. In total, 24 cox1 haplotypes of E. granulosus s.s. were identified including the already described variants: Eg01 (in 6 samples), Eg33 (in 4 samples), EgCl04 (in 2 samples), Eg03 (in 1 sample) and Eg32 (in 1 sample). From the twenty-five other isolates of E. granulosus s.s. a total of 19 non-previously described cox1 haplotypes were identified and named as EgKyr1 to EgKyr19. The most common haplotype infecting human is the EgKyr1 which was found in 5 isolates.The cob/nad2/cox1 A2 haplotype of E. multilocularis is responsible for the majority of human infections in Kyrgyzstan and is also found in the majority of dogs included in this study. Further similar studies in different parts of Asia could elucidate if it is also the most common variant infecting humans in other countries. It remains unknown if this particular haplotype presents differences in virulence which could have contributed to the emergency of alveolar echinococcosis in Kyrgyzstan. In the case of E. granulosus s.s. it seems that there is no dominant haplotype infecting humans in Kyrgzstan. Further characterization of biological or antigenic features of dominant mitochondrial haplotypes could help to elucidate if they present differences which could be relevant in the diagnostic, pathogenicity or in the host/parasite interaction when infecting humans.


Assuntos
Doenças Transmissíveis Emergentes/parasitologia , Equinococose/parasitologia , Echinococcus granulosus/classificação , Echinococcus granulosus/genética , Echinococcus multilocularis/classificação , Echinococcus multilocularis/genética , Variação Genética , Adulto , Animais , Análise por Conglomerados , Doenças Transmissíveis Emergentes/epidemiologia , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Equinococose/epidemiologia , Equinococose/veterinária , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Haplótipos , Proteínas de Helminto/genética , Humanos , Incidência , Quirguistão/epidemiologia , Masculino , Tipagem de Sequências Multilocus , NADH Desidrogenase/genética
2.
J Med Microbiol ; 69(5): 689-696, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375980

RESUMO

Introduction. Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) has various properties, including anti-inflammatory, antioxidant and anticancer activities. However, the mechanism underlying the role of rhein in antimicrobial activity remains largely unknown.Aim. This study aims to identify potential natural compounds of rhein that are capable of inhibiting Cutibacterium acnes and elucidate the effects of rhein on NADH dehydrogenase-2 activity in C. acnes.Methodology. The anti-C. acnes activity of compounds was analysed using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the paper disc diffusion test and the checkerboard dilution test. To check whether rhein was inhibitory, putative type II NADH dehydrogenase (NDH-2) of C. acnes was analysed, cloned and expressed in Escherichia coli, and then NDH-2 purification was assessed with Ni-NTA before rhein inhibition of NADH dehydrogenase-2 activity was checked with ferricyanide [K3Fe(CN)6] as a substrate.Results. The results showed that the MIC of rhein against C. acnes was 6.25 µg ml-1, while the MBC was 12.5 µg ml-1, and there was a 38 mm inhibition zone in the paper disc diffusion test. Rhein showed an additive two- to fourfold reduction of the MIC value with four antibiotics on the checkerboard dilution test. The purified NADH dehydrogenase gene product showed a size of approximately 51 kDa and had a V max of 23 µmol and a K m of 280 µm. The inhibitory effect of rhein against NADH dehydrogenase-2 activity was non-competitive with ferricyanide [K3Fe(CN)6] with a K i value of 3.5-4.5 µm.Conclusion. This study provided evidence of the inhibitory effects of rhein on the growth of C. acnes by blocking of NADH dehydrogenase-2 activity. This mechanism of inhibitory activity in the reduction of ROS formation and ATP productivity should be further tested in C. acnes and the question of whether rhein inhibits the natural growth of C. acnes should be investigated.


Assuntos
Antraquinonas/farmacologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Infecções por Bactérias Gram-Positivas/microbiologia , NADH Desidrogenase/antagonistas & inibidores , Propionibacterium acnes/efeitos dos fármacos , Propionibacterium acnes/enzimologia , Antraquinonas/uso terapêutico , Antibacterianos/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Cinética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Propionibacterium acnes/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes
3.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(2): 159-167, 2020 Apr 24.
Artigo em Chinês | MEDLINE | ID: mdl-32458605

RESUMO

OBJECTIVE: To characterize a species of the genus Tricula and parasitized trematodes in schistosomiasis-endemic areas of Yunnan Province using a molecular analysis, so as to understand their taxonomic positions. METHODS: Tricula spp. and Oncomelania snails were collected from Xiangyun County, Yunnan Province, and cercaria parasitizing snails were observed using crushing followed by microscopy. Cercaria parasitizing Tricula snails at various morphologies were sampled using a shedding method. Genomic DNA was extracted from snail soft tissues and cercariae, and the 16S rRNA, COI, 28S rDNA genes in snails and the ND1 and 28S rDNA genes in cercariae were amplified using a PCR assay and sequenced. The species of Tricula snails and their parasitized trematodes was characterized using sequence alignment and phylogenetic analysis. RESULTS: Among 382 Tricula snails detected, there were three types of trematode cercariae found, including the non-forked (20.94%, 80/382), double-forked (3.40%, 13/382) and swallow shapes (7.07%, 27/382). Sequence and phylogenetic analyses showed that the 16S rRNA, COI and 28S rDNA gene sequences of this species of Tricula had high homology to those in Delavaya dianchiensis, and were clustered in a branch. Sequencing analysis of the ND1 and 28S rDNA genes revealed that the non-forked cercariae belonged to the family Pleu- rogenidae, the swallow-shaped cercariae belonged to the family Opecoelidae, and the double-forked cercariae belonged to another species of the genus Schistosoma that was different from S. sinensium and S. ovuncatum. CONCLUSIONS: The species and taxonomy of Triculla spp. and their parasitized trematodes are preliminarily determined in schistosomiasis-endemic areas of Yunnan Province; however, further studies are required to investigate the more definite taxonomy and pathogenicity.


Assuntos
Cercárias , Filogenia , Caramujos/classificação , Caramujos/parasitologia , Trematódeos , Animais , Cercárias/genética , Cercárias/isolamento & purificação , China , Complexo IV da Cadeia de Transporte de Elétrons/genética , Doenças Endêmicas , NADH Desidrogenase/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 28S/genética , Esquistossomose/epidemiologia , Esquistossomose/parasitologia , Caramujos/genética , Trematódeos/genética , Trematódeos/isolamento & purificação
4.
Food Chem ; 322: 126759, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32283374

RESUMO

Detection of animal species in meat product is crucial to prevent adulterated and unnecessary contamination during processing. Gold standard is the real-time PCR assays, which can be conducted at highly equipped laboratories. Toward the development of point-of-need test, two rapid molecular assays based on recombinase polymerase amplification (RPA) for the detection of pork and horse DNA were established. Target genes are the porcine mitochondrial ND2 and equine ATP 6-8 genes. The pork and horse_RPA assays detected 16 and one DNA molecules/µl in eleven to six minutes, respectively. The myoglobin in the meat did not influence the assays performances, while the presence of high background-DNA induced a one log decrease in the sensitivity. Both assays are highly specific and identify down to 0.1% of their target DNA in meat mixtures. Both RPA assays could be used on-site as a rapid and mobile detection system to determine contamination of meat products.


Assuntos
DNA/análise , Cavalos/genética , Carne/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Suínos/genética , Animais , DNA/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , NADH Desidrogenase/genética , Sistemas Automatizados de Assistência Junto ao Leito , Recombinases/metabolismo
5.
PLoS One ; 15(4): e0231965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324772

RESUMO

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that forms part of the normal human microbiota and it is also an opportunistic pathogen, responsible for 30% of all nosocomial urinary tract infections. P. aeruginosa carries a highly branched respiratory chain that allows the colonization of many environments, such as the urinary tract, catheters and other medical devices. P. aeruginosa respiratory chain contains three different NADH dehydrogenases (complex I, NQR and NDH-2), whose physiologic roles have not been elucidated, and up to five terminal oxidases: three cytochrome c oxidases (COx), a cytochrome bo3 oxidase (CYO) and a cyanide-insensitive cytochrome bd-like oxidase (CIO). In this work, we studied the composition of the respiratory chain of P. aeruginosa cells cultured in Luria Broth (LB) and modified artificial urine media (mAUM), to understand the metabolic adaptations of this microorganism to the growth in urine. Our results show that the COx oxidases play major roles in mAUM, while P. aeruginosa relies on CYO when growing in LB medium. Moreover, our data demonstrate that the proton-pumping NQR complex is the main NADH dehydrogenase in both LB and mAUM. This enzyme is resistant to HQNO, an inhibitory molecule produced by P. aeruginosa, and may provide an advantage against the natural antibacterial agents produced by this organism. This work offers a clear picture of the composition of this pathogen's aerobic respiratory chain and the main roles that NQR and terminal oxidases play in urine, which is essential to understand its physiology and could be used to develop new antibiotics against this notorious multidrug-resistant microorganism.


Assuntos
Materiais Biomiméticos , Meios de Cultura , Oxirredutases/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Urina , Aerobiose , Transporte de Elétrons , NADH Desidrogenase/metabolismo , Quinonas/metabolismo
6.
Gene ; 746: 144638, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244054

RESUMO

Most chloroplast genes in Zanthoxylum schinifolium (Korean pepper) and Z. piperitum (Japanese pepper) are subject to neutral or purifying selection (Ka/Ks values < 1); however, NAD(P)H dehydrogenase subunit G (ndhG) has a Ka/Ks value of 1.43, which may indicate positive selection. Here, we modeled the ZsNdhG and ZpNdhG structures by comparing them with the NuoJ subunit of respiratory complex I in Escherichia coli, revealing the locations of four amino acid differences between ZsNdhG and ZpNdhG. As these polymorphisms were located at the end of a membrane-spanning α-helix or in peptide loops external to the membrane, they are not expected to have major effects on the membrane-embedding properties of these proteins. However, we found that C-to-U RNA editing occurred at the ndhG-50 sites of ndhG (uCa to uUa, Ser to Leu) in both species, resulting in changes to an amino acid located in the middle of a membrane-spanning α-helix, which may maintain its hydrophobicity. RNA editing at the ndhG-50 site was conserved in many plant species, and the modeled structures of Anthoceros formosae NdhG and Spirodela polyrhiza NdhB provided further evidence that RNA editing increases the hydrophobicity of membrane-embedded proteins. We also speculate that the polar residues inside membrane-spanning α-helices serve to support the protein structure. This report represents the first RNA-editing site identified in Zanthoxylum and points to the importance of considering RNA editing when identifying positively selected genes based on Ka/Ks values.


Assuntos
Proteínas de Cloroplastos/genética , NADH Desidrogenase/genética , Polimorfismo Genético , Edição de RNA , Zanthoxylum/genética , Especificidade da Espécie
7.
Mitochondrial DNA A DNA Mapp Seq Anal ; 31(3): 120-130, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32212876

RESUMO

To understand the genetic diversity and dispersal history of Schlegel's Japanese gecko (Gekko japonicus), we performed genetic analyses and paleo-species distributional modelling. For the genetic analysis, we analyzed mitochondrial DNA (mtDNA) (cytochrome b [Cytb] and NADH dehydrogenase 2 [ND2]) and seven microsatellite loci of 353 individuals from 11 populations (2 east coast China, 4 west and central coast Japan and 5 Korea). For the paleo-species distribution modelling, we used 432 occurrence data points (125 China, 291 Japan and 16 Korea) over the Pleistocene and Holocene. China is inferred to be the source population, which had higher genetic diversity (mtDNA) and more private alleles (mtDNA) compared to Japanese and Korean populations. Differences between the three counties were very small in the mtDNA haplotype network despite some genetic structure among the three countries. Microsatellite analysis inferred that genetic exchange has actively occurred among the Chinese, Japanese and Korean populations. Suitable habitats in Japan should have been plentiful by the mid-Holocene, but have only recently become available in Korea. These results suggest that dispersal of G. japonicus occurred after the Holocene warming from the east coast of China to the west and central coasts of Japan and Korea, and gene flow is actively occurring among the three countries.


Assuntos
Citocromos b/genética , Lagartos/genética , NADH Desidrogenase/genética , Alelos , Animais , China , DNA Mitocondrial/genética , Ecossistema , Genes Mitocondriais/genética , Variação Genética/genética , Genética Populacional/métodos , Genoma Mitocondrial/genética , Haplótipos/genética , Japão , Repetições de Microssatélites/genética , Mitocôndrias/genética , Filogenia , Filogeografia/métodos , República da Coreia , Análise de Sequência de DNA/métodos
8.
Parasitol Res ; 119(5): 1697-1701, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32125518

RESUMO

The worldwide occurring common liver fluke Fasciola hepatica can infect humans and animals and leads to considerable illness and economic loss annually. The aim of this study was to determine the genetic diversity of F. hepatica in Austria. In total, 31 adult flukes isolated from cattle from various regions in Austria were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene sequences. It was shown that Austrian isolates of F. hepatica reveal extensive genetic diversity. To the best of our knowledge, these are the first data on the diversity of F. hepatica in Austria.


Assuntos
Doenças dos Bovinos/parasitologia , DNA de Helmintos/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Fasciola hepatica/genética , Fasciolíase/veterinária , NADH Desidrogenase/genética , Animais , Áustria , Bovinos , DNA Mitocondrial/genética , Variação Genética/genética , Haplótipos , Humanos , Filogenia , Reação em Cadeia da Polimerase
9.
Genes (Basel) ; 11(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936803

RESUMO

Understanding mitochondrial DNA (mtDNA) evolution and inheritance has broad implications for animal speciation and human disease models. However, few natural models exist that can simultaneously represent mtDNA transmission bias, mutation, and copy number variation. Certain isolates of the nematode Caenorhabditis briggsae harbor large, naturally-occurring mtDNA deletions of several hundred basepairs affecting the NADH dehydrogenase subunit 5 (nduo-5) gene that can be functionally detrimental. These deletion variants can behave as selfish DNA elements under genetic drift conditions, but whether all of these large deletion variants are transmitted in the same preferential manner remains unclear. In addition, the degree to which transgenerational mtDNA evolution profiles are shared between isolates that differ in their propensity to accumulate the nduo-5 deletion is also unclear. We address these knowledge gaps by experimentally bottlenecking two isolates of C. briggsae with different nduo-5 deletion frequencies for up to 50 generations and performing total DNA sequencing to identify mtDNA variation. We observed multiple mutation profile differences and similarities between C. briggsae isolates, a potentially species-specific pattern of copy number dysregulation, and some evidence for genetic hitchhiking in the deletion-bearing isolate. Our results further support C. briggsae as a practical model for characterizing naturally-occurring mtgenome variation and contribute to the understanding of how mtgenome variation persists in animal populations and how it presents in mitochondrial disease states.


Assuntos
Proteínas de Bactérias/genética , Caenorhabditis/genética , Genoma Mitocondrial/genética , NADH Desidrogenase/genética , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Deleção de Genes , Variação Genética/genética , Mitocôndrias/genética , Mutação/genética , NADH Desidrogenase/metabolismo , Filogenia , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA/métodos , Deleção de Sequência/genética
10.
Parasitol Res ; 119(3): 985-990, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31953565

RESUMO

The present paper deals with the post-mortem diagnostics of onchocerciasis and the molecular detection of causative agents of this disease in wild ruminant ungulates (Cervus elaphus, Dama dama and Capreolus capreolus). The animals were shot in hunting seasons 2017 and 2018, in two regions of the Eastern Slovakia. The total number of examined skins was fifty-eight. The presence of subcutaneous nodules was confirmed in 27.59% (95% CI 16-39) of animals. All positive skins belonged to red deer individuals (47.06%; 95% CI 30-64). The nodules were present mainly in the back area and in the lumbar area, and their sizes ranged from 2.9 to 24.1 mm, with the average count of 10 nodules per animal. Thirteen worms, isolated from the nodules collected from 13 animals, were subjected to molecular identification. Applying the standard PCR method, targeting the mitochondrial 12S rRNA, 16S rRNA and NADH-dehydrogenase gene, and subsequent sequencing, all the worms were identified as Onchocerca flexuosa Wedl, 1856. The sequences were submitted to GenBank under specific accession numbers. Two samples were identified as Onchocerca flexuosa haplotype B, in which T176A and A177T were present. Despite the presence of mutations in the 12S rRNA of the Onchocerca flexuosa, the standardized PCR remains to be a very specific and sensitive method that uses this fragment as a selectable marker for the detection of the studied parasite.


Assuntos
Cervos/parasitologia , Onchocerca/isolamento & purificação , Oncocercose/veterinária , Animais , NADH Desidrogenase/genética , Onchocerca/classificação , Onchocerca/genética , Oncocercose/parasitologia , RNA Ribossômico/genética , RNA Ribossômico 16S/genética , Pele/parasitologia , Eslováquia
11.
PLoS One ; 15(1): e0227991, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990943

RESUMO

This study reports complete plastome sequences for six species of Neotropical Cranichideae and focuses on identification of the most variable regions (hotspots) in this group of orchids. These structure of these six plastomes is relatively conserved, exhibiting lengths ranging between 142,599 to 154,562 bp with 36.7% GC on average and exhibiting typical quadripartite arrangement (LSC, SSC and two IRs). Variation detected in the LSC/IR and SSC/IR junctions is explained by the loss of ndhF and ycf1 length variation. For the two genera of epiphytic clade in Spiranthinae, almost whole sets of the ndh-gene family were missing. Eight mutation hotspots were identified based on nucleotide diversity, sequence variability and parsimony-informative sites. Three of them (rps16-trnQ, trnT-trnL, rpl32-trnL) seem to be universal hotspots in the family, and the other five (trnG-trnR, trnR-atpA, trnP-psaJ, rpl32-infA, and rps15-ycf1) are described for the first time as orchid molecular hotspots. These regions have much more variation than all those used previously in phylogenetics of the group and offer useful plastid markers for phylogenetic, barcoding and population genetic studies. The use of whole plastomes or exclusive no-gap matrices also positioned with high support the holomycotrophic Rhizanthella among Orchidoideae plastomes in model-based analyses, showing the utility of plastomes for phylogenetic placement of this unusual genus.


Assuntos
Regulação da Expressão Gênica de Plantas , Variação Genética , Genoma , Orchidaceae/genética , Filogenia , Plastídeos/genética , Composição de Bases , Brasil , Mapeamento Cromossômico , Código de Barras de DNA Taxonômico/métodos , Ontologia Genética , Anotação de Sequência Molecular , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Orchidaceae/classificação , Orchidaceae/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
12.
Parasitol Res ; 119(2): 411-421, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31915912

RESUMO

Varroa destructor, a parasitic mite of the western honey bee, Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. Population genetics studies of the mite have provided information on two mitochondrial haplotypes infecting honey bee colonies, named K and J (after Korea and Japan, respectively, where they were originally identified). On the American continent, the K haplotype is much more prevalent, with the J haplotype only detected in some areas of Brazil. The aims of the present study were to assess the genetic diversity of V. destructor populations in the major beekeeping region of Argentina and to evaluate the presence of heteroplasmy at the nucleotide level. Phoretic mites were collected from managed A. mellifera colonies in ten localities, and four mitochondrial DNA (mtDNA) regions (COXI, ND4, ND4L, and ND5) were analyzed. Based on cytochrome oxidase subunit I (COXI) sequencing, exclusively the K haplotype of V. destructor was detected. Furthermore, two sub-haplotypes (KArg-N1 and KArg-N2) were identified from a variation in ND4 sequences and the frequency of these sub-haplotypes was found to significantly correlate with geographical latitude. The occurrence of site heteroplasmy was also evident for this gene. Therefore, ND4 appears to be a sensitive marker for detecting genetic variability in mite populations. Site heteroplasmy emerges as a phenomenon that could be relatively frequent in V. destructor.


Assuntos
Abelhas/parasitologia , DNA Mitocondrial/genética , Variação Genética/genética , Proteínas Mitocondriais/genética , Varroidae/genética , Animais , Argentina , Criação de Abelhas , Brasil , Complexo I de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Haplótipos , Japão , NADH Desidrogenase/genética , República da Coreia
13.
Mol Cell ; 77(1): 189-202.e6, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31668496

RESUMO

The proteolytic turnover of mitochondrial proteins is poorly understood. Here, we used a combination of dynamic isotope labeling and mass spectrometry to gain a global overview of mitochondrial protein turnover in yeast cells. Intriguingly, we found an exceptionally high turnover of the NADH dehydrogenase, Nde1. This homolog of the mammalian apoptosis inducing factor, AIF, forms two distinct topomers in mitochondria, one residing in the intermembrane space while the other spans the outer membrane and is exposed to the cytosol. The surface-exposed topomer triggers cell death in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1; however, it is strongly enriched in respiratory-deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised cells from growing populations.


Assuntos
Morte Celular/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Proteostase/fisiologia , Proteases Dependentes de ATP/metabolismo , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Citosol/metabolismo , Transporte de Elétrons/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Am J Hum Genet ; 106(1): 92-101, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31866046

RESUMO

Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Fibroblastos/patologia , Doença de Leigh/etiologia , Doenças Mitocondriais/etiologia , Proteínas Mitocondriais/genética , Mutação , NADH Desidrogenase/genética , Alelos , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Doença de Leigh/patologia , Masculino , Doenças Mitocondriais/patologia , Linhagem , Fenótipo
15.
Anal Chim Acta ; 1095: 219-225, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31864626

RESUMO

Herein we present a general and turn-on strategy for enzymatic bioassays on the basis of redox state dependent emission of gold nanoclusters (AuNCs). The photoluminescence of AuNCs was quenched obviously by the oxidative ferricyanide while unaffected by its corresponding reduced state, i.e., ferrocyanide. The distinctive quenching abilities for AuNCs by the redox couple (ferricyanide/ferrocyanide) enabled their utility as new fluorescent sensing platforms to detect redox-related phenomena. The proposed protocols were conducted by using the model oxidoreductases of glucose oxidase (GOx) and the enzyme cascade of lactate dehydrogenase (LDH)/diaphorase to catalytically convert ferricyanide to ferrocyanide, which switched on fluorescence of the detection systems. The detection limit for glucose and lactate was found to be as low as 0.12 and 0.09 µM, respectively. This work features the first use of the redox couple of ferricyanide/ferrocyanide in fluorescent bioanalysis, which enables versatile, signal on and highly sensitive/selective detections as compared to the state of the art fluorescently enzymatic sensing platforms. Importantly, considering the significance of ferricyanide/ferrocyanide involves in numerous other oxidoreductases mediated biocatalysis, this protocol has wide versatility that enables combination with oxidoreductases related reactions for biosensing.


Assuntos
Corantes Fluorescentes/química , Glucose/análise , Ácido Láctico/análise , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/métodos , Animais , Bovinos , Ferricianetos/química , Fluorescência , Glucose/química , Glucose Oxidase/química , Ouro/química , Humanos , L-Lactato Desidrogenase/química , Ácido Láctico/química , Limite de Detecção , NADH Desidrogenase/química , Oxirredução , Soroalbumina Bovina/química
16.
BMC Res Notes ; 12(1): 800, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31823814

RESUMO

OBJECTIVE: Analyze key features of the anglerfish Lophius piscatorius mitochondrial transcriptome based on high-throughput total RNA sequencing. RESULTS: We determined the complete mitochondrial DNA and corresponding transcriptome sequences of L. piscatorius. Key features include highly abundant mitochondrial ribosomal RNAs (10-100 times that of mRNAs), and that cytochrome oxidase mRNAs appeared > 5 times more abundant than both NADH dehydrogenase and ATPase mRNAs. Unusual for a vertebrate mitochondrial mRNA, the polyadenylated COI mRNA was found to harbor a 75 nucleotide 3' untranslated region. The mitochondrial genome expressed several non-canonical genes, including the long noncoding RNAs lncCR-H, lncCR-L and lncCOI. Whereas lncCR-H and lncCR-L mapped to opposite strands in a non-overlapping organization within the control region, lncCOI appeared novel among vertebrates. We found lncCOI to be a highly abundant mitochondrial RNA in antisense to the COI mRNA. Finally, we present the coding potential of a humanin-like peptide within the large subunit ribosomal RNA.


Assuntos
Peixes/genética , Mitocôndrias/genética , Transcriptoma/genética , Adenosina Trifosfatases/genética , Animais , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Peixes/metabolismo , Genoma Mitocondrial , Sequenciamento de Nucleotídeos em Larga Escala , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/metabolismo , NADH Desidrogenase/genética , Filogenia , RNA Antissenso/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mitocondrial/genética , RNA Ribossômico/genética
17.
Genes (Basel) ; 10(12)2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810328

RESUMO

Renal cell carcinoma (RCC) is associated with various genetic alterations. Although whole-genome/exome sequencing analysis has revealed that nuclear genome alterations are associated with clinical outcomes, the association between nucleotide alterations in the mitochondrial genome and RCC clinical outcomes remains unclear. In this study, we analyzed somatic mutations in the mitochondrial D-loop region, using RCC samples from 61 consecutive patients with localized RCC. Moreover, we analyzed the relationship between D-loop mutations and NADH dehydrogenase subunit 1 (MT-ND1) mutations, which we previously found to be associated with clinical outcomes in localized RCC. Among the 61 localized RCCs, 34 patients (55.7%) had at least one mitochondrial D-loop mutation. The number of D-loop mutations was associated with larger tumor diameter (> 32 mm) and higher nuclear grade (≥ ISUP grade 3). Moreover, patients with D-loop mutations showed no differences in cancer-specific survival when compared with patients without D-loop mutations. However, the co-occurrence of D-loop and MT-ND1 mutations improved the predictive accuracy of cancer-related deaths among our cohort, increasing the concordance index (C-index) from 0.757 to 0.810. Thus, we found that D-loop mutations are associated with adverse pathological features in localized RCC and may improve predictive accuracy for cancer-specific deaths when combined with MT-ND1 mutations.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Mutação , NADH Desidrogenase/genética , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Masculino , Pessoa de Meia-Idade , NADH Desidrogenase/metabolismo , Proteínas de Neoplasias/metabolismo , Valor Preditivo dos Testes , Taxa de Sobrevida
18.
PLoS Negl Trop Dis ; 13(12): e0007887, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31869329

RESUMO

Myiasis is the infestation of human tissues by dipterous fly larvae of the class Insecta. Clogmia albipunctatus, family Psychodidae, is one of the most medically important insects that cause human myiasis. The aim of the present study is the morphological identification and the molecular characterization of moth flies causing many cases of urinary myiasis in Egypt, based on sequencing of the mitochondrial DNA of the larvae. Seven urinary samples of patients complaining of urinary symptoms and giving a history of low socioeconomic level were examined. Recovered larvae were identified using light microscopy and SEM. For molecular identification, the mitochondrial genes Cytochrome B (cytB), NADH1, NADH1, and 16S were sequenced and phylogenetically analyzed. The morphological and molecular characterization could accurately diagnose our patients to have C. albipunctatus infestation. Such results provided the initial set of data on the molecular identification and phylogenetic analysis of moth flies based on DNA barcoding in Egypt.


Assuntos
Larva/anatomia & histologia , Larva/genética , Miíase/parasitologia , Psychodidae/anatomia & histologia , Psychodidae/genética , Doenças Urológicas/parasitologia , Adolescente , Animais , Criança , Pré-Escolar , Análise por Conglomerados , Citocromos b/genética , Código de Barras de DNA Taxonômico , DNA Mitocondrial/química , DNA Mitocondrial/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Egito , Feminino , Humanos , Larva/classificação , Masculino , Microscopia , Microscopia Eletrônica de Varredura , NADH Desidrogenase/genética , Filogenia , Psychodidae/classificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Adulto Jovem
19.
Nat Commun ; 10(1): 4970, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672993

RESUMO

The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb's respiratory chain, and highlight the challenges associated with targeting the pathogen's respiratory enzymes for tuberculosis drug development.


Assuntos
Antituberculosos/uso terapêutico , Desenvolvimento de Medicamentos , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mycobacterium tuberculosis/genética , NADH Desidrogenase/genética , Tuberculose/tratamento farmacológico , Adaptação Fisiológica/genética , Animais , Callithrix , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Imidazóis/farmacologia , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , NADH Desidrogenase/antagonistas & inibidores , Piperidinas/farmacologia , Piridinas/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/patologia
20.
Parasit Vectors ; 12(1): 520, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690347

RESUMO

BACKGROUND: Cysticercosis caused by the metacestode larval stage of Taenia hydatigena is a disease of veterinary and economic importance. A considerable level of genetic variation among isolates of different intermediate hosts and locations has been documented. Generally, data on the genetic population structure of T. hydatigena is scanty and lacking in Nigeria. Meanwhile, similar findings in other cestodes like Echinococcus spp. have been found to be of epidemiological importance. Our aim, therefore, was to characterize and compare the genetic diversity of T. hydatigena population in Nigeria based on three mitochondrial DNA markers as well as to assess the phylogenetic relationship with populations from other geographical regions. METHODS: In the present study, we described the genetic variation and diversity of T. hydatigena isolates from Nigerian sheep and goats using three full-length mitochondrial genes: the cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), and NADH dehydrogenase subunit 5 (nad5). RESULTS: The median-joining network of concatenated cox1-nad1-nad5 sequences indicated that T. hydatigena metacestodes of sheep origin were genetically distinct from those obtained in goats and this was supported by high FST values of nad1, cox1, and concatenated cox1-nad1-nad5 sequences. Genetic variation was also found to be higher in isolates from goats than from sheep. CONCLUSIONS: To the best of our knowledge, the present study described the genetic variation of T. hydatigena population for the first time in Nigeria using full-length mitochondrial genes and suggests the existence of host-specific variants. The population indices of the different DNA markers suggest that analysis of long mitochondrial DNA fragments may provide more information on the molecular ecology of T. hydatigena. We recommend that future studies employ long mitochondrial DNA sequence in order to provide reliable data that would explain the extent of genetic variation in different hosts/locations and the biological and epidemiological significance.


Assuntos
Genes Mitocondriais , Doenças das Cabras/parasitologia , Doenças dos Ovinos/parasitologia , Taenia/genética , Teníase/veterinária , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Marcadores Genéticos , Variação Genética , Cabras , NADH Desidrogenase/genética , Nigéria , Filogenia , Ovinos , Taenia/classificação , Taenia/isolamento & purificação , Teníase/parasitologia
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