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1.
Chem Biol Interact ; 311: 108796, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421116

RESUMO

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5'-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1-3 and 5'-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin. Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.


Assuntos
Plaquetas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nitrilos/farmacologia , Nucleotidases/genética , Nucleosídeos de Purina/metabolismo , Piretrinas/farmacologia , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrólise , Fígado/enzimologia , Fígado/metabolismo , Masculino , Espectrometria de Massas , Nitrilos/sangue , Nitrilos/metabolismo , Nucleotidases/metabolismo , Piretrinas/sangue , Piretrinas/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Ratos Wistar
2.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 5-11, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102351

RESUMO

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-ß-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -ß-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS: Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.


Assuntos
Pirofosfatases/química , Canais de Cátion TRPM/química , Canais de Cátion TRPM/isolamento & purificação , Escherichia coli/genética , Glucosídeos/química , Humanos , Domínios Proteicos , Estabilidade Proteica , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombina/metabolismo
3.
Nat Chem Biol ; 15(6): 575-582, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101919

RESUMO

We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Å resolution provides insights into the molecular basis of the deNADding activity in the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells, and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism.


Assuntos
NAD/metabolismo , Pirofosfatases/metabolismo , RNA Mensageiro/metabolismo , Humanos , NAD/química , Pirofosfatases/química , Pirofosfatases/genética , RNA Mensageiro/química
4.
Org Lett ; 21(9): 3218-3222, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30995052

RESUMO

An Escherichia coli strain was constructed for the efficient import of nicotinamide adenine dinucleotide (NAD) analogues into cells by limiting extracellular degradation while expressing an efficient NAD importer. In vivo functions of three NAD analogues were characterized. Nicotinamide hypoxanthine dinucleotide was identified as an inhibitor of NAD synthesis. Nicotinamide cytosine dinucleotide had excellent biocompatibility and was used for characterizing a growth-dependent degradation of in vivo nicotinamide cofactors.


Assuntos
Escherichia coli/metabolismo , NAD/análogos & derivados , NAD/metabolismo , Niacinamida/química , Coenzimas/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Citosina/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Estrutura Molecular , Mutação , NAD/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo
5.
Virus Res ; 266: 43-47, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30951792

RESUMO

In 1936, John Joseph Bittner identified mouse mammary tumor virus (MMTV), a milk transmitted beta retrovirus, a form of single-stranded positive-sense RNA virus. A retrovirus inserts a copy of its genome into the DNA of a host cell, thus altering the cell's genome. In the current analysis, we searched for MMTV sequences within the human genome. To compare the MMTV genome to the human genome, we used BLAT, the Blast-Like Alignment Tool of the UCSC Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. 60 MMTV sequences were in the human genome. Of 56 sequences from the MMTV POL gene, 36 POL sequences were from the same part of the gene, beginning at viral nucleotide 4800 but of different lengths. 8 viral sequences began at nucleotide ∼3430 of the POL gene. Four viral sequences were from GAGdUTPase, encoded by the MMTV PRO gene. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is an enzyme present in several major retroviral families. In MMTV dUTPase may be essential for viral replication. Since BLAT identified no MMTV envelope (env) sequence in the human genome, the env sequences from breast tumors and normal breast tissue found in other studies may have come from an MMTV infection. However, no one is certain how MMTV could enter human cells, since the cells do not have a cellular receptor for MMTV, as do mouse cells.


Assuntos
Neoplasias da Mama/virologia , Mama/virologia , Genes env/genética , Vírus do Tumor Mamário do Camundongo/genética , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Bases de Dados Genéticas , Feminino , Genes pol/genética , Genoma Humano/genética , Humanos , Pirofosfatases/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Software
6.
Medicine (Baltimore) ; 98(15): e15065, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30985656

RESUMO

Extensive coronary calcification without significant stenosis, described as calcific coronary artery disease (CCAD) may cause abnormal myocardial perfusion and hence generalized ischemia. There is a discrepancy in the expression pattern of CCAD compared to the well-known atherosclerotic disease which raises questions about the exact pathophysiology of coronary calcification and whether there is a genetic etiology for it.In this pilot study we studied 3 candidate genes, ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), ATP Binding Cassette Subfamily C Member 6 (ABCC6), and 5'-Nucleotidase Ecto (NT5E) involved in pyrophosphate (PPi) and inorganic phosphate (Pi) metabolism, which may predispose to coronary arterial or valvular calcification. We studied 70 patients with calcific cardiac disease; 65 with CCAD (age 43-83 years) and 5 with calcific aortic valve disease (CAVD) (age 76-82 years).Five DNA variants potentially affecting protein function were found in 6 patients. One variant is a known disease-causing mutation in the ABCC6 gene. Our findings support that disturbances in the PPi and Pi metabolism might influence the development of CCAD and CAVD. However, segregation in the families must first be performed to ascertain any damaging effect of these variants we have found.We report 4 new genetic variants potentially related to coronary calcification, through the disturbed Pi and PPi metabolism. The search for direct causative genetic variants in coronary artery and aortic valve calcification must be broadened with other genes particularly those involved with Pi and PPi metabolism.


Assuntos
5'-Nucleotidase/genética , Calcinose/genética , Variação Genética , Cardiopatias/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Proteínas Ligadas por GPI/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Fatores Sexuais , Suécia , Rigidez Vascular/genética
7.
PLoS Genet ; 15(3): e1007605, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30856165

RESUMO

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.


Assuntos
Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/genética , Catarata/enzimologia , Catarata/genética , Hipogonadismo/enzimologia , Hipogonadismo/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Pirofosfatases/deficiência , Animais , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Homozigoto , Humanos , Inosina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/enzimologia , Mutação , Linhagem , Pirofosfatases/genética , RNA/genética , RNA/metabolismo , Sequenciamento Completo do Exoma
8.
Asian Pac J Cancer Prev ; 20(3): 801-808, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30909691

RESUMO

Background: Nudix Hydrolase 15 gene (NUDT15) encodes nucleotide triphosphate diphosphatase which metabolizes the purine analog drugs, such as anticancer thiopurine and anti-gout allopurinol. Genetic variants on Nudix Hydrolase 15 gene (NUDT15) gene effects the drug's hydrolyses and hence increases the susceptibility to drug-induced toxicity. The NUDT15 gene has been genotyped in various ethnic groups, however, it has not been genotyped among the Middle Eastern Arab Jordanian population. Aim: The current study aimed to identify NUDT15 genetic variants among Jordanian Arab population. Method: The DNA samples were isolated from leukocytes of 85 unrelated Jordanian Arab volunteers. The coding regions of NUDT15 gene; Exon 1,2 and 3, in addition to some regions of intron 1,2 and 3'UTR, were amplified using polymerase chain reaction (PCR). the PCR products were then subjected to purification and sequenced using Applied Biosystems Model (ABI3730x1). Results: Six NUDT15 genetic variants were found among the volunteers.The results were as followed: A novel synonymous variant 36A>G on exon 1 (6%, 95%CI= 3- 9%), the intronic IVS1 +116C>T variant on intron 1 (0.6%, 95%CI= 0-2%), the non-synonymous variant on exon 3; 415C>T (0.6%, 95%CI= 0-2%), A novel non-synonymous variant on exon 3; 404C>A (0.6%, 95%CI= 0-2%) , and two novel variants on 3'UTR ;502G>A (2%, 95%CI= 0.5-4%) and 588T>C (0.6%, 95%CI= 0-2%). NUDT15 36A>G wasfound to be the most common allele among Jordanians was. In silico softwares predicted that the novel NUDT15 404C>A was harmful and affected NUDT15 enzyme'sstability and function. Furthermore, the frequency of NUDT15 IVS1 +116C>T , among Jordanians, showed to be significantly lower from what was reported in other ethnicities with ap value > 0.05 on the other hand, the frequency of 415C>T variant showed to be similar to Europeans in contrast to Asians and Indians that showed to be significantly lower (p value > 0.05). Conclusions: The frequency of NUDT15 genetic variants is low among the Jordanian volunteers and significantly lower than other ethnic groups. The findings of this study may increase our understanding of the inter-individual variation in the response to purine analog drugs. Further clinical studies are needed to investigate the influence of novel NUDT15 404C>A on drug metabolism and response.


Assuntos
Árabes/genética , Grupos Étnicos/genética , Variação Genética , Genética Populacional , Pirofosfatases/genética , Adulto , Éxons , Feminino , Genótipo , Humanos , Masculino
9.
EBioMedicine ; 41: 427-442, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827930

RESUMO

BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.


Assuntos
Retrovirus Endógenos/genética , Epigenômica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Redutases do Citocromo/genética , Retrovirus Endógenos/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas/genética , Pirofosfatases/genética , Taxa de Sobrevida , Sequências Repetidas Terminais/genética , Enzimas de Conjugação de Ubiquitina/genética
10.
Emerg Microbes Infect ; 8(1): 40-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866758

RESUMO

The ability of Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.


Assuntos
Perfilação da Expressão Gênica/métodos , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Nucleotídeos de Pirimidina/química , Pirofosfatases/genética , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Quimioterapia Combinada , Regulação Bacteriana da Expressão Gênica , Humanos , Ferro/metabolismo , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Pirimidinas/metabolismo , Pirofosfatases/metabolismo , Análise de Sequência de RNA/métodos , Células THP-1
11.
JAMA ; 321(8): 773-785, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30806694

RESUMO

Importance: Use of thiopurines may be limited by myelosuppression. TPMT pharmacogenetic testing identifies only 25% of at-risk patients of European ancestry. Among patients of East Asian ancestry, NUDT15 variants are associated with thiopurine-induced myelosuppression (TIM). Objective: To identify genetic variants associated with TIM among patients of European ancestry with inflammatory bowel disease (IBD). Design, Setting, and Participants: Case-control study of 491 patients affected by TIM and 679 thiopurine-tolerant unaffected patients who were recruited from 89 international sites between March 2012 and November 2015. Genome-wide association studies (GWAS) and exome-wide association studies (EWAS) were conducted in patients of European ancestry. The replication cohort comprised 73 patients affected by TIM and 840 thiopurine-tolerant unaffected patients. Exposures: Genetic variants associated with TIM. Main Outcomes and Measures: Thiopurine-induced myelosuppression, defined as a decline in absolute white blood cell count to 2.5 × 109/L or less or a decline in absolute neutrophil cell count to 1.0 × 109/L or less leading to a dose reduction or drug withdrawal. Results: Among 1077 patients (398 affected and 679 unaffected; median age at IBD diagnosis, 31.0 years [interquartile range, 21.2 to 44.1 years]; 540 [50%] women; 602 [56%] diagnosed as having Crohn disease), 919 (311 affected and 608 unaffected) were included in the GWAS analysis and 961 (328 affected and 633 unaffected) in the EWAS analysis. The GWAS analysis confirmed association of TPMT (chromosome 6, rs11969064) with TIM (30.5% [95/311] affected vs 16.4% [100/608] unaffected patients; odds ratio [OR], 2.3 [95% CI, 1.7 to 3.1], P = 5.2 × 10-9). The EWAS analysis demonstrated an association with an in-frame deletion in NUDT15 (chromosome 13, rs746071566) and TIM (5.8% [19/328] affected vs 0.2% [1/633] unaffected patients; OR, 38.2 [95% CI, 5.1 to 286.1], P = 1.3 × 10-8), which was replicated in a different cohort (2.7% [2/73] affected vs 0.2% [2/840] unaffected patients; OR, 11.8 [95% CI, 1.6 to 85.0], P = .03). Carriage of any of 3 coding NUDT15 variants was associated with an increased risk (OR, 27.3 [95% CI, 9.3 to 116.7], P = 1.1 × 10-7) of TIM, independent of TPMT genotype and thiopurine dose. Conclusions and Relevance: Among patients of European ancestry with IBD, variants in NUDT15 were associated with increased risk of TIM. These findings suggest that NUDT15 genotyping may be considered prior to initiation of thiopurine therapy; however, further study including additional validation in independent cohorts is required.


Assuntos
Colite Ulcerativa/genética , Doença de Crohn/genética , Metiltransferases/metabolismo , Pirofosfatases/genética , Adolescente , Adulto , Estudos de Casos e Controles , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Doença de Crohn/tratamento farmacológico , Doença de Crohn/metabolismo , Grupo com Ancestrais do Continente Europeu , Exoma , Feminino , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Contagem de Leucócitos , Masculino , Metiltransferases/genética , Metiltransferases/uso terapêutico , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Adulto Jovem
12.
Biochim Biophys Acta Proteins Proteom ; 1867(4): 376-381, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639426

RESUMO

Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.


Assuntos
Nucleotídeos de Desoxiguanina/química , Pirofosfatases/química , Pirofosfatases/genética , Domínio Catalítico , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Temperatura Ambiente , Termolisina/química
13.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(1): 100-104, 2019 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-30675872

RESUMO

As an important drug during maintenance treatment of acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) has several side effects, including hepatotoxicity and bone marrow suppression. Since its tolerability varies from person to person, 6-MP treatment should be individualized. The deficiency of thiopurine methyltransferase (TPMT) enzyme activity is associated with 6-MP intolerance. There is a lower frequency of mutation in TPMT alleles among Asian patients. Recent studies have shown that in ALL patients with NUDT15 gene mutation, the maximum tolerated dose of 6-MP is lower than the conventional dose. The article reviews the significance of NUDT15 gene in individualized treatment with 6-MP in children with ALL.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Pirofosfatases/genética , Antimetabólitos Antineoplásicos , Criança , Humanos , Mercaptopurina , Metiltransferases
14.
Transplant Proc ; 50(10): 3925-3927, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30577288

RESUMO

BACKGROUND: Azathioprine (AZA) is the drug recommended for the continuation of immunosuppressive treatment after renal transplant in women during pregnancy. CASE REPORT: A 37-year-old Japanese female developed agranulocytosis and severe alopecia after initiation of AZA (50 mg), used as an alternative to mycophenolate mofetil (MMF, 1000 mg) therapy in anticipation of a planned pregnancy. Within 4 days of the initiation of AZA therapy, the patient developed a high fever, leucopenia, and cranial alopecia. Genetic testing revealed a homozygous polymorphism of NUDT15 (rs116855232, NM_018283.3:c.415C>T: p.Arg139Cys), which has previously been identified as a risk factor for AZA-related complications in patients with inflammatory bowel disease. CONCLUSION: Genetic screening for NUDT15 could contribute to the prevention of serious adverse reactions to AZA and provide the opportunity for personalized medicine. Identification of a safe alternative to MMF during pregnancy after a renal transplant is a problem to be resolved in the future.


Assuntos
Azatioprina/efeitos adversos , Imunossupressores/efeitos adversos , Transplante de Rim , Pirofosfatases/genética , Adulto , Agranulocitose/induzido quimicamente , Alopecia/induzido quimicamente , Feminino , Rejeição de Enxerto/prevenção & controle , Homozigoto , Humanos , Transplante de Rim/métodos , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Fatores de Risco
15.
Wiad Lek ; 71(4): 815-820, 2018.
Artigo em Russo | MEDLINE | ID: mdl-30099416

RESUMO

OBJECTIVE: Introduction: More than 100 genes have been described associations between single nucleotide polymorphisms and type 2 diabetes mellitus (T2DM). Among these candidate genes, Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1), is located on the long arm of chromosome 6 (6q23.2) and encodes for a protein which is one of the factors determining the insulin sensitivity. An allelic polymorphism in exon 4 of ENPP1 (rs1044498) has been designated K121Q and widely investigated in T2DM in different populations. The aim: To analyze the association between ENPP1 K121Q polymorphism with the risk factors of type 2 diabetes mellitus in the Ukrainian population. PATIENTS AND METHODS: Materials and methods: Venous blood of 317 patients with type 2 diabetes mellitus and 302 controls was used for analysis. ENPP1 K121Q genotyping was performed using PCR-RFLP method. RESULTS: Results: Our results revealed that ratio of K/K homozygotes, K/Q heterozygotes and Q/Q homozygotes between case and control groups was significantly different (59.3%, 34.1%, 6.6% vs 67.9%, 28.5%, 3.6%, P = 0.05). Method of binary logistic regression shown that a reliable relationship was established in the general group for KQ/QQ vs K/K genetic model (P = 0.027). It was shown that in carriers of the minor Q-allele, the risk of T2DM is 1.4x higher than in homozygotes in the main K-allele (95% CI = 1.043-2.016). After adjusting for age, sex, smoking habit, BMI, obesity and the presence of hypertension, the reliability of these results persisted (P = 0.026). CONCLUSION: Conclusions: ENPP1 K121Q polymorphism is associated with T2DM in Ukrainian population. In carriers of the minor Q-allele the risk of T2DM is 1.4x higher than in homozygotes in the main K-allele. The risk increases in patients with BMI ≥ 25 kg/m2.


Assuntos
Diabetes Mellitus Tipo 2/genética , Grupo com Ancestrais do Continente Europeu/genética , Diester Fosfórico Hidrolases/genética , Polimorfismo Genético/genética , Polimorfismo de Fragmento de Restrição , Pirofosfatases/genética , Adulto , Alelos , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/genética , Polimorfismo de Nucleotídeo Único , Ucrânia
16.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30045981

RESUMO

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Mutagênese , Pirofosfatases/genética , Ribavirina/farmacologia , Antivirais/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Nucleotídeos/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Ribavirina/metabolismo , Transdução de Sinais
17.
Gene ; 675: 225-232, 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29958952

RESUMO

In the current study, a sample population of Northern Iranians was selected to investigate the association of K121Q, rs1799774, rs7754561, and rs997509 ENPP1 gene variants and their haplotypes with T2DM. Genomic DNAs of 978 samples were extracted by Salting Out standard technique and then genotyped by the TaqMan assay. The results show significant differences between study groups for K121Q (p = 0.0004) under a Dominant and rs7754561 (p = 0.002) under a co-dominant hereditary model. Based on allele frequency, there was a significant difference between two study groups at K121Q and rs7754561 variants (p = 0.010 and p = 0.01, respectively). There was no evidence for an association between ENPP1 haplotypes and overall risk of T2DM. Genotype-phenotype sub-analyses showed no significant relationship of four studied polymorphisms with age, gender, FBS, and systolic and diastolic blood pressures. Homology modeling and molecular docking of ENPP1 in K173 and Q173 models with ATP, AMP, and 2'3'-cGAMP as ligands revealed that all ligands had a more binding affinity to Lys173 protein model, and 2'3'-cGAMP had a higher affinity to both ENPP1 protein models compared to ATP and AMP. These findings suggest that ENPP1 gene variants may have a potential impact on the occurrence of T2DM in Northern Iranians.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Polimorfismo de Nucleotídeo Único , Pirofosfatases/química , Pirofosfatases/genética , Adulto , Idoso , Substituição de Aminoácidos , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo
18.
Medicine (Baltimore) ; 97(27): e11236, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29979387

RESUMO

BACKGROUND: Previous studies suggested an association between K121Q (rs1044498 C > A) in ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene and the risk of coronary heart disease (CHD), but the results have been inconsistent. In this study, we performed a meta-analysis of several trials to systematically summarize their potential association. METHODS: Relevant articles were identified by searching electronic databases for studies published prior to March 2018. We carefully reviewed published studies on ENPP1 genetic polymorphism in relation to CHD susceptibility. The data extracted from selected high-quality studies were analyzed using STATA statistical software (StataCorp LP, College Station, TX, USA). RESULTS: Nine eligible studies which contained a combined total of 1547 CHD cases and 2213 healthy controls were chosen in the present meta-analysis. Our results indicated that K121Q strongly correlated with increased risk of CHD. The subgroup analysis on race, sample source, disease type, sex, age, and genotype showed that in Caucasians, K121Q strongly correlated with increased risk of CHD, but no difference was found in Chinese. Both single factor and multiple factor regression showed that race, sample origin, disease type, sex, age, and genotype were not the source of heterogeneity. CONCLUSIONS: Our meta-analysis revealed that the K121Q (rs1044498 C > A) in the ENPP1 gene is a risk factor for CHD.


Assuntos
Doença das Coronárias/genética , Diester Fosfórico Hidrolases/genética , Polimorfismo de Nucleotídeo Único/genética , Pirofosfatases/genética , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Doença das Coronárias/etnologia , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco
19.
Proc Natl Acad Sci U S A ; 115(29): E6845-E6854, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29915072

RESUMO

Bacteria respond to nutritional stresses by changing the cellular concentration of the alarmone (p)ppGpp. This control mechanism, called the stringent response, depends on two enzymes, the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT in Escherichia coli and related bacteria. Because SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in these bacteria, SpoT activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which is lethal. To date, however, no such regulation of SpoT (p)ppGpp hydrolase activity has been documented in E. coli In this study, we show that Rsd directly interacts with SpoT and stimulates its (p)ppGpp hydrolase activity. Dephosphorylated HPr, but not phosphorylated HPr, of the phosphoenolpyruvate-dependent sugar phosphotransferase system could antagonize the stimulatory effect of Rsd on SpoT (p)ppGpp hydrolase activity. Thus, we suggest that Rsd is a carbon source-dependent regulator of the stringent response in E. coli.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Pirofosfatases/metabolismo , Proteínas Repressoras/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Proteínas Repressoras/genética
20.
Eur J Histochem ; 62(2): 2877, 2018 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-29943954

RESUMO

In dorsal root ganglion (DRG) neurons, ATP is an important neurotransmitter in nociceptive signaling through P2 receptors (P2Rs) such as P2X2/3R, and adenosine is also involved in anti-nociceptive signaling through adenosine A1R. Thus, the clearance system for adenine nucleotide/nucleoside plays a critical role in regulation of nociceptive signaling, but there is little information on it, especially ectoenzyme expression profiles in DRG. In this study, we examined expression and localization of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs), by which ATP is metabolized to AMP, in rat DRG. The mRNA expression levels of ENPP2 were greater than those of ENPP1 and ENPP3 in rat DRGs. On immunohistochemical analysis, ENPP1, 2 and 3 were found in soma of DRG neurons. Immunopositive rate of ENPP3 was greater than that of ENPP1 and ENPP2 in all DRG neurons. ENPP3, as compared with ENPP1 and ENPP2, was expressed mainly by isolectin B4-positive cells, and slightly by neurofilament 200-positive ones. In this way, the expression profile of ENPP1, 2 and 3 was different in DRGs, and they were mainly expressed in small/medium-sized DRG neurons. Moreover, ENPP1-, 2- and 3-immunoreactivities were colocalized with P2X2R, P2X3R and prostatic acid phosphatase (PAP), as an ectoenzyme for metabolism from AMP to adenosine. Additionally, PAP-immunoreactivity was colocalized with equilibrative nucleoside transporter (ENT) 1, as an adenosine uptake system. These results suggest that the clearance system consisted of ENPPs, PAP and ENT1 plays an important role in regulation of nociceptive signaling in sensory neurons.


Assuntos
Gânglios Espinais/metabolismo , Imuno-Histoquímica/métodos , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Células Receptoras Sensoriais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Masculino , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Ratos , Ratos Sprague-Dawley
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