RESUMO
The present study aims to analyze the effect of apricot kernels' extract (AKE) and amygdalin (AMY) on bleomycin-induced genetic alternations. Five endpoints were analyzed: cell survival, Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus, reverse point mutations in ilv1-92 allele, and mitotic crossing-over in the ade2 locus. The present work provides the first experimental evidence that bleomycin induces Ty1 retrotransposition in Saccharomyces cerevisiae. New data is obtained that the degree of DNA protection of AMY and AKE depends on the studied genetic event. AKE has been found to provide significant protection against bleomycin-induced Ty1 retrotransposition due to better-expressed antioxidant potential. On the other side, AMY better-expressed protection against bleomycin-induced mitotic gene conversion and reverse mutations may be attributed to the activation of the repair enzymes.
Assuntos
Amigdalina , Prunus armeniaca , Proteínas de Saccharomyces cerevisiae , Alelos , Amigdalina/farmacologia , Bleomicina/farmacologia , Conversão Gênica , Extratos Vegetais/farmacologia , Mutação Puntual , Prunus armeniaca/genética , Prunus armeniaca/metabolismo , Retroelementos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Nuclear-encoded Atp23 was previously shown to have dual functions, including processing the yeast Atp6 precursor and assisting the assembly of yeast mitochondrial ATP synthase. However, it remains unknown whether there are genes functionally complementary to ATP23 to rescue atp23 null mutant. In the present paper, we screen and characterize three revertants of atp23 null mutant and reveal a T1121G point mutation in the mitochondrial gene COX1 coding sequence, which leads to Val374Gly mutation in Cox1, the suppressor in the revertants. This was verified further by the partial restoration of mitochondrial ATP synthase assembly in atp23 null mutant transformed with exogenous hybrid COX1 T1121G mutant plasmid. The predicted tertiary structure of the Cox1 p.Val374Gly mutation showed no obvious difference from wild-type Cox1. By further chase labeling with isotope [35S]-methionine, we found that the stability of Atp6 of ATP synthase increased in the revertants compared with the atp23 null mutant. Taking all the data together, we revealed that the T1121G point mutation of mitochondrial gene COX1 could partially restore the unassembly of mitochondrial ATP synthase in atp23 null mutant by increasing the stability of Atp6. Therefore, this study uncovers a gene that is partially functionally complementary to ATP23 to rescue ATP23 deficiency, broadening our understanding of the relationship between yeast the cytochrome c oxidase complex and mitochondrial ATP synthase complex.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Mitocondriais/genética , Metaloproteases/genética , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação Puntual/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , DNA Mitocondrial/genética , Mutação com Perda de Função/genéticaRESUMO
Glutathione peroxidase 4 (GPX4), as the only enzyme in mammals capable of reducing esterified phospholipid hydroperoxides within a cellular context, protects cells from ferroptosis. We identified a homozygous point mutation in the GPX4 gene, resulting in an R152H coding mutation, in three patients with Sedaghatian-type spondylometaphyseal dysplasia. Using structure-based analyses and cell models, including patient fibroblasts, of this variant, we found that the missense variant destabilized a critical loop, which disrupted the active site and caused a substantial loss of enzymatic function. We also found that the R152H variant of GPX4 is less susceptible to degradation, revealing the degradation mechanism of the GPX4 protein. Proof-of-concept therapeutic treatments, which overcome the impaired R152H GPX4 activity, including selenium supplementation, selective antioxidants and a deuterated polyunsaturated fatty acid were identified. In addition to revealing a general approach to investigating rare genetic diseases, we demonstrate the biochemical foundations of therapeutic strategies targeting GPX4.
Assuntos
Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Medicina de Precisão , Humanos , Mutação Puntual , Estudo de Prova de ConceitoRESUMO
A protein's function is closely related to its structural properties. Mutations can affect the functionality of a protein. Different cancer tissues have found disordered expression of the cyclin-dependent kinase 2-associated Protein 1 (CDK2AP1) gene. A protein molecule's conformational flexibility affects its interaction with phytochemicals and their biological partners at various levels. Boerhavia diffusa has been investigated most extensively for its medicinal activities like anticancer properties. It contains many bioactive compounds like Boeravinone A, Boeravinone B, Boeravinone C, Boeravinone D, Boeravinone E, Boeravinone F, Boeravinone G, Boeravinone H, Boeravinone I and Boeravinone J. We have studied to analyse the binding efficacy properties as well as essential dynamic behaviour, free energy landscape of both the native and mutant protein CDK2AP1 with bioactive compounds from Boerhavia diffusa plant extracts through computational approaches by homology modelling, docking and molecular dynamics simulation. From the molecular docking study, we found that. Boeravinone J have best binding affinity (-7.9 kcal/mol) towards the native protein of CDKAP1 compared to others phytochemicals. However, we found the binding energy for H23R and C105R (mutation point) -7.8 and -7.6 kcal/mol, respectively. A single minima energy point (from 100 ns molecular dynamics simulation study) was found in the H23R mutant with Boeravinone J complex suggested that minimum structural changes with less conformational mobility compared C105A mutant model.Communicated by Ramaswamy H. Sarma.
Assuntos
Nyctaginaceae , Mutação Puntual , Quinase 2 Dependente de Ciclina/genética , Simulação de Acoplamento Molecular , Proteínas Mutantes , Nyctaginaceae/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.
Assuntos
Adenosina Desaminase/fisiologia , Agamaglobulinemia/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Neutrófilos/citologia , Imunodeficiência Combinada Severa/imunologia , Adenosina Desaminase/genética , Células Cultivadas , Corpos Embrioides/citologia , Fibroblastos/enzimologia , Granulócitos/citologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Hidroxiureia/farmacologia , Lactente , Masculino , Mutação de Sentido Incorreto , Mielopoese , Niacinamida/farmacologia , Mutação Puntual , Ribonucleotídeo Redutases/antagonistas & inibidoresRESUMO
Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.
Assuntos
Brassinosteroides/metabolismo , Flores/anatomia & histologia , Flores/genética , Genes de Plantas , Loci Gênicos , Polinização/genética , Turnera/genética , Turnera/metabolismo , Alelos , Arabidopsis/genética , Brassinosteroides/farmacologia , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Genótipo , Germinação/efeitos dos fármacos , Germinação/genética , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas , Mutação Puntual , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Polinização/efeitos dos fármacos , Esteroides Heterocíclicos/farmacologia , Turnera/crescimento & desenvolvimentoRESUMO
Growth hormone (GH) is one of the critical factors in maintaining glucose metabolism. B-cell translocation gene 2 (BTG2) and yin yang 1 (YY1) are key regulators of diverse metabolic processes. In this study, we investigated the link between GH and BTG2-YY1 signaling pathway in glucose metabolism. GH treatment elevated the expression of hepatic Btg2 and Yy1 in primary mouse hepatocytes and mouse livers. Glucose production in primary mouse hepatocytes and serum blood glucose levels were increased during GH exposure. Overexpression of hepatic Btg2 and Yy1 induced key gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6 phosphatase (G6PC) as well as glucose production in primary mouse hepatocytes, whereas this phenomenon was markedly diminished by knockdown of Btg2 and Yy1. Here, we identified the YY1-binding site on the Pck1 and G6pc gene promoters using reporter assays and point mutation analysis. The regulation of hepatic gluconeogenic genes induced by GH treatment was clearly linked with YY1 recruitment on gluconeogenic gene promoters. Overall, this study demonstrates that BTG2 and YY1 are novel regulators of GH-dependent regulation of hepatic gluconeogenic genes and glucose production. BTG2 and YY1 may be crucial therapeutic targets to intervene in metabolic dysfunction in response to the GH-dependent signaling pathway.
Assuntos
Gluconeogênese/genética , Hormônio do Crescimento/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular , Glucose/biossíntese , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Hormônio do Crescimento/administração & dosagem , Hepatócitos , Humanos , Injeções Intraperitoneais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Modelos Animais , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Mutação Puntual , Cultura Primária de Células , Regiões Promotoras Genéticas , Transdução de Sinais/genéticaRESUMO
Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders characterised by deficits in social interactions and repetitive behaviours. ASDs have a strong genetic basis with mutations involved in the development and function of neural circuitry. Shank proteins act as master regulators of excitatory glutamatergic synapses, and Shank mutations have been identified in people with ASD. Here, we have investigated the impact of ASD-associated Shank2 single nucleotide variants (SNVs) at the synaptic level, and the potential of in vitro zinc supplementation to prevent synaptic deficits. Dissociated rat hippocampal cultures expressing enhanced green fluorescent protein (EGFP) tagged Shank2-Wildtype (WT), and ASD-associated Shank2 single nucleotide variants (SNVs: S557N, V717F, and L1722P), were cultured in the absence or presence of 10 µM zinc. In comparison to Shank2-WT, ASD-associated Shank2 SNVs induced significant decreases in synaptic density and reduced the frequency of miniature excitatory postsynaptic currents. These structural and functional ASD-associated synaptic deficits were prevented by chronic zinc supplementation and further support zinc supplementation as a therapeutic target in ASD.
Assuntos
Transtorno do Espectro Autista/genética , Suplementos Nutricionais , Hipocampo/patologia , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Mutação Puntual/genética , Sinapses/patologia , Zinco/farmacologia , Animais , Animais Recém-Nascidos , Transtorno do Espectro Autista/patologia , Feminino , Ácido Glutâmico/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Sinapses/efeitos dos fármacosRESUMO
The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.
Assuntos
Fusão de Membrana/efeitos dos fármacos , Proteínas SNARE/antagonistas & inibidores , alfa-Sinucleína/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Dopamina/metabolismo , Dopamina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Lipossomos , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/farmacologia , Mutação Puntual , Ligação Proteica , Multimerização Proteica , Proteolipídeos/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas SNARE/fisiologia , Proteína 2 Associada à Membrana da Vesícula/antagonistas & inibidores , Proteína 2 Associada à Membrana da Vesícula/fisiologia , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadeRESUMO
ABSTRACT: Tailored therapy based on dual priming oligonucleotide-based polymerase chain reaction (DPO-PCR) can be considered an alternative to overcome the low eradication rate in high clarithromycin-resistance areas. The triple therapy (TT) duration of the tailored approach in most studies was 7âdays for patients without point mutation. However, recent western guidelines have recommended a treatment duration of 14âdays. The aim of this study was to compare the success rate of 7 and 14âdays of TT for eradicating Helicobacter pylori without point mutation, as determined by DPO-PCR.Between Feb 2016 and Feb 2019, medical records of patients who underwent DPO-PCR were reviewed. Patients without point mutation as determined by DPO-PCR were enrolled in this study. The eradication success rate and adverse events were evaluated.A total of 366 patients without A2142G and A2143G point mutation were enrolled. The success rates of 7-day and 14-day TT were 88.4% (168/190) and 85.9% (151/176) by intention to treat analysis (Pâ=â.453) and 90.8% (168/185) and 90.4% (151/167) by per-protocol analysis (Pâ=â.900), respectively. The adverse event rates showed no significant difference between the 2 groups.In patients without point mutation based on DPO-PCR results, 7-day TT is as effective as 14-day TT. Therefore, 7âdays may be considered as a cost-effective treatment duration in Korea.
Assuntos
Antibacterianos/administração & dosagem , Infecções por Helicobacter/tratamento farmacológico , Antibacterianos/efeitos adversos , Antibacterianos/economia , Análise Custo-Benefício , Esquema de Medicação , Farmacorresistência Bacteriana , Quimioterapia Combinada , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação Puntual , RNA Ribossômico 23S/genética , República da CoreiaRESUMO
The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
Assuntos
Endossomos/efeitos dos fármacos , Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Lisossomos/efeitos dos fármacos , Doença de Parkinson/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Endossomos/fisiologia , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lisossomos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Mutação Puntual , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Distribuição Aleatória , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.
Assuntos
Ácidos Graxos Ômega-3/administração & dosagem , Hipertrigliceridemia/dietoterapia , Triglicerídeos/metabolismo , Amidoidrolases/deficiência , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Bile/metabolismo , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/análogos & derivados , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Humanos , Hipertrigliceridemia/metabolismo , Hipolipemiantes/administração & dosagem , Hipolipemiantes/metabolismo , Absorção Intestinal/efeitos dos fármacos , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação Puntual , Taurina/análogos & derivados , Taurina/metabolismoAssuntos
ADP Ribose Transferases/genética , Códon sem Sentido , Proteínas de Membrana/genética , ADP Ribose Transferases/imunologia , Adolescente , Alelos , Tipagem e Reações Cruzadas Sanguíneas , DNA Complementar/genética , Feminino , Mutação da Fase de Leitura , Hemofilia A/complicações , Humanos , Isoanticorpos/sangue , Articulação do Joelho/cirurgia , Masculino , Proteínas de Membrana/imunologia , Pais , Fenótipo , Mutação Puntual , Deleção de Sequência , SíriaRESUMO
RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the RPE65 gene causing inherited retinal dystrophies, most notably Leber congenital amaurosis (LCA) and autosomal recessive retinitis pigmentosa (arRP), which are both severe and early onset disease entities. We previously reported a mutation, D477G, co-segregating with the disease in a late-onset form of autosomal dominant RP (adRP) with choroidal involvement; uniquely, it is the only RPE65 variant to be described with a dominant component. Families or individuals with this variant have been encountered in five countries, and a number of subsequent studies have been reported in which the molecular biological and physiological properties of the variant have been studied in further detail, including observations of possible novel functions in addition to reduced RPE65 enzymatic activity. With regard to the latter, a human phase 1b proof-of-concept study has recently been reported in which aspects of remaining vision were improved for up to one year in four of five patients with advanced disease receiving a single one-week oral dose of 9-cis retinaldehyde, which is the first report showing efficacy and safety of an oral therapy for a dominant form of RP. Here, we review data accrued from published studies investigating molecular mechanisms of this unique variant and include hitherto unpublished material on the clinical spectrum of disease encountered in patients with the D477G variant, which, in many cases bears striking similarities to choroideremia.
Assuntos
Substituição de Aminoácidos , Genes Dominantes , Mutação de Sentido Incorreto , Mutação Puntual , Retinose Pigmentar/genética , cis-trans-Isomerases/genética , Idade de Início , Animais , Coroideremia , Ensaios Clínicos Fase I como Assunto , DNA Complementar/administração & dosagem , DNA Complementar/genética , Terapia de Reposição de Enzimas , Feminino , Técnicas de Introdução de Genes , Terapia Genética , Vetores Genéticos/uso terapêutico , Humanos , Amaurose Congênita de Leber/enzimologia , Amaurose Congênita de Leber/genética , Masculino , Camundongos , Linhagem , Estudo de Prova de Conceito , Isoformas de Proteínas/genética , Retinaldeído/uso terapêutico , Retinose Pigmentar/diagnóstico por imagem , Retinose Pigmentar/enzimologia , Retinose Pigmentar/terapia , cis-trans-Isomerases/deficiência , cis-trans-Isomerases/fisiologia , cis-trans-Isomerases/uso terapêuticoRESUMO
Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material.
Assuntos
Modelos Animais de Doenças , Técnicas de Genotipagem/métodos , Técnicas de Diagnóstico Molecular/métodos , Distrofia Muscular de Duchenne/genética , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Animais , Birrefringência , Avaliação Pré-Clínica de Medicamentos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Sensibilidade e Especificidade , Peixe-ZebraRESUMO
Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene arise in 25-30% of all acute myeloid leukemia (AML) patients. These mutations lead to constitutive activation of the protein product and are divided in two broad types: internal tandem duplication (ITD) of the juxtamembrane domain (25% of cases) and point mutations in the tyrosine kinase domain (TKD). Patients with FLT3 ITD mutations have a high relapse risk and inferior cure rates, whereas the role of FLT3 TKD mutations still remains to be clarified. Additionally, growing research indicates that FLT3 status evolves through a disease continuum (clonal evolution), where AML cases can acquire FLT3 mutations at relapse - not present in the moment of diagnosis. Several FLT3 inhibitors have been tested in patients with FLT3-mutated AML. These drugs exhibit different kinase inhibitory profiles, pharmacokinetics and adverse events. First-generation multi-kinase inhibitors (sorafenib, midostaurin, lestaurtinib) are characterized by a broad-spectrum of drug targets, whereas second-generation inhibitors (quizartinib, crenolanib, gilteritinib) show more potent and specific FLT3 inhibition, and are thereby accompanied by less toxic effects. Notwithstanding, all FLT3 inhibitors face primary and acquired mechanisms of resistance, and therefore the combinations with other drugs (standard chemotherapy, hypomethylating agents, checkpoint inhibitors) and its application in different clinical settings (upfront therapy, maintenance, relapsed or refractory disease) are under study in a myriad of clinical trials. This review focuses on the role of FLT3 mutations in AML, pharmacological features of FLT3 inhibitors, known mechanisms of drug resistance and accumulated evidence for the use of FLT3 inhibitors in different clinical settings.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Sorafenibe/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Compostos de Anilina/farmacologia , Benzimidazóis/farmacologia , Benzotiazóis/farmacologia , Carbazóis/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Previsões , Furanos , Transplante de Células-Tronco Hematopoéticas , Humanos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Quimioterapia de Manutenção/métodos , Mutação , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Mutação Puntual , Pirazinas/farmacologia , Piridazinas/farmacologia , Recidiva , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.
Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/uso terapêutico , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Substituição de Aminoácidos , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Criança , Ciprofloxacina/uso terapêutico , Análise Mutacional de DNA/métodos , Diarreia/diagnóstico , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Humanos , Isoleucina/genética , Testes de Sensibilidade Microbiana , Peru , Treonina/genéticaRESUMO
Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum-inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.
Assuntos
Antibacterianos/farmacologia , Boratos/farmacologia , Dickeya/efeitos dos fármacos , Pectobacterium/efeitos dos fármacos , Solanum tuberosum/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dickeya/genética , Dickeya/crescimento & desenvolvimento , Dickeya/fisiologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Teste de Complementação Genética , Movimento , Pectobacterium/genética , Pectobacterium/crescimento & desenvolvimento , Pectobacterium/fisiologia , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Doenças das Plantas/microbiologia , Mutação Puntual , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismoRESUMO
OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.
Assuntos
Aspergilose/microbiologia , Aspergillus flavus/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Voriconazol/uso terapêutico , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/genética , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/isolamento & purificação , Biomarcadores/análise , Sistema Enzimático do Citocromo P-450/genética , Análise Mutacional de DNA/métodos , Regulação Fúngica da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Técnicas de Tipagem Micológica , Mutação Puntual , Esterol 14-Desmetilase/genéticaRESUMO
BACKGROUND: Oral antibiotics, such as ciprofloxacin (CFX), are widely used for the treatment of acute and chronic pouchitis. Most bacterial mutations that confer quinolone resistance are at Ser-83 and Asp-87 in the gyrA gene and Ser-80 and Glu-84 in the parC gene. METHODS: We obtained 51 stool samples from 43 patients who were diagnosed with ulcerative colitis and underwent ileal pouch-anal anastomosis. Patients were divided into 2 groups: 13 patients with CFX treatment of pouchitis and 30 patients without pouchitis. After extraction of fecal DNA, the amount of Escherichia coli 16S rRNA, gyrA, and parC gene DNA were measured using real-time polymerase chain reaction (PCR). Possible mutations at gyrA 83 and 87 and at parC 80 and 84 were investigated by PCR cloning and sequencing, and mutation rates were quantified by rapid PCR-restriction fragment length polymorphism. RESULTS: Samples from both CFX-treated and -untreated patients had comparable levels of gyrA and parC gene DNA. Nucleic acid and amino acid mutations were identified at gyrA 83 and 87, and at parC 80 and 84. We successfully quantified mutation rates at gyrA 83 and 87, and at parC 84, all of which were significantly higher in samples from CFX-treated patients (70, 84, and 38%) than from CFX-untreated patients (13, 11, and 5%). CONCLUSION: E. coli in patient pouches may have mutations in their gyrA and parC genes that produce CFX resistance. Mutation rates of these genes were significantly higher in samples from CFX-treated patients. This study contributes to understanding the decrease and loss of CFX effectiveness against pouchitis.