RESUMO
Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder ß-thalassemia, mutations in the ß-globin gene (HBB) lead to a reduction in the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by Unc-51-like autophagy activating kinase 1 (ULK1)-dependent autophagy and that stimulating this pathway by systemic mammalian target of rapamycin complex 1 (mTORC1) inhibition alleviates ß-thalassemia pathologies. We show here that disrupting the bicistronic microRNA gene miR-144/451 alleviates ß-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through 2 mechanisms. Loss of miR-451 upregulated its target messenger RNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor adenosine monophosphate-activated protein kinase (AMPK). The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. In addition, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1, causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates, and improve hematological indices in ß-thalassemia. The beneficial effects of miR-144/451 loss in ß-thalassemia were inhibited by the disruption of Cab39 or Ulk1 genes. Together, our findings link the severity of ß-thalassemia to a highly expressed erythroid microRNA locus and a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.
Assuntos
MicroRNAs , Talassemia beta , Humanos , Talassemia beta/terapia , Proteínas Quinases Ativadas por AMP/metabolismo , alfa-Globinas , Autofagia/genética , MicroRNAs/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.
Assuntos
Transtornos da Insuficiência da Medula Óssea/patologia , Mutação com Ganho de Função , Heterozigoto , Síndromes Mielodisplásicas/patologia , Proteína de Replicação A/genética , Encurtamento do Telômero , Telômero/genética , Adolescente , Adulto , Transtornos da Insuficiência da Medula Óssea/etiologia , Transtornos da Insuficiência da Medula Óssea/metabolismo , Diferenciação Celular , Criança , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/metabolismo , Adulto JovemRESUMO
The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack.
Assuntos
Arabidopsis , Phaseolus , Arabidopsis/genética , Arabidopsis/metabolismo , Phaseolus/genética , Phaseolus/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Pseudomonas syringae/fisiologia , Pectinas/metabolismo , Parede Celular/metabolismoRESUMO
The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pró-Fármacos/farmacologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Pirimidinas/farmacologia , Ativação Metabólica , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína/metabolismo , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Drogas em Investigação/química , Drogas em Investigação/metabolismo , Drogas em Investigação/farmacologia , Deleção de Genes , Conformação Molecular , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Oxirredução , Filogenia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Pirimidinas/química , Pirimidinas/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
We describe here the identification and functional characterization of the enzyme O-GlcNAcase (OGA) as an RNA polymerase II elongation factor. Using in vitro transcription elongation assays, we show that OGA activity is required for elongation in a crude nuclear extract system, whereas in a purified system devoid of OGA the addition of rOGA inhibited elongation. Furthermore, OGA is physically associated with the known RNA polymerase II (pol II) pausing/elongation factors SPT5 and TRIM28-KAP1-TIF1ß, and a purified OGA-SPT5-TIF1ß complex has elongation properties. Lastly, ChIP-seq experiments show that OGA maps to the transcriptional start site/5' ends of genes, showing considerable overlap with RNA pol II, SPT5, TRIM28-KAP1-TIF1ß, and O-GlcNAc itself. These data all point to OGA as a component of the RNA pol II elongation machinery regulating elongation genome-wide. Our results add a novel and unexpected dimension to the regulation of elongation by the insertion of O-GlcNAc cycling into the pol II elongation regulatory dynamics.
Assuntos
Antígenos de Neoplasias/química , Histona Acetiltransferases/química , Hialuronoglucosaminidase/química , Proteínas Nucleares/química , RNA Polimerase II/química , Proteínas Repressoras/química , Fatores de Elongação da Transcrição/química , Antígenos de Neoplasias/metabolismo , Histona Acetiltransferases/metabolismo , Humanos , Hialuronoglucosaminidase/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Proteínas Repressoras/metabolismo , Elongação da Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/metabolismo , Proteína 28 com Motivo TripartidoRESUMO
Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (AsV) to arsenite (AsIII) by arsenate reductases, which is then extruded by arsenite permeases. One of these Gram-positive bacteria, Corynebacterium glutamicum, the workhorse of biotechnological research, is also resistant to arsenic. To sanitize contaminated soils and waters, C. glutamicum strains were modified to work as arsenic "biocontainers." Two chromosomally encoded ars operons (ars1 and ars2) are responsible for As resistance. The genes within these operons encode for metalloregulatory proteins (ArsR1/R2), arsenite permeases (Acr3-1/-2), and arsenate reductases (ArsC1/C2/C1'). ArsC1/C2 arsenate reductases are coupled to the low molecular weight thiol mycothiol (MSH) and to the recently discovered mycoredoxin-1 (Mrx-1) present in most Actinobacteria. This MSH/Mrx-1 redox system protects cells against different forms of stress, including reactive oxygen species (ROS), metals, and antibiotics. ROS can modify functional sulfur cysteines by oxidizing the thiol (-SH) to a sulfenic acid (-SOH). These oxidation-sensitive protein cysteine thiols are redox regulated by the MSH/Mrx-1 couple in Corynebacterium and Mycobacterium. In summary, the molecular mechanisms involved in arsenic resistance system in C. glutamicum have paved the way for understanding the cellular response against oxidative stress in Actinobacteria.
Assuntos
Arsênio/metabolismo , Corynebacterium glutamicum/metabolismo , Arsênio/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica , Óperon , OxirreduçãoRESUMO
BACKGROUND: Breast cancer is the most deadly malignancy in Mexican women. Although treatment has improved, it may significantly affect bone mineral status in those who receive it. The aim of this study was to assess the impact of cancer treatment on bone mineral density (BMD) and bone mineral content (BMC), in patients with breast cancer and explore the interaction of menopausal status and clinical stage with cancer treatment on such changes. METHODS: A quasi-experimental design was applied with measurements before and after a chemotherapy treatment in 40 patients with primary diagnosis of invasive breast cancer. BMD and body composition measurements were taken by dual X-ray absorptiometry (DXA) and changes in these variables due to therapy were analyzed using mixed regression for repeated measurements. RESULTS: Significant loss was found in femoral neck and L2-L4 BMD (p < 0.001). Patients diagnosed with osteopenia or osteoporosis received calcium + vitamin D supplementation (600 mg/200 IU day). It showed a protective effect in the decrease of femoral neck BMD and total BMC. BMD loss in both femoral neck and L2-L4 BMD was higher in premenopausal women: 0.023 g/cm2 in femoral neck and 0.063 g/cm2 in L2-L4 (p < 0.001), while in postmenopausal women BMD loss was 0.015 g/cm2 in femoral neck and 0.035 g/cm2 in L2-L4 (p = 0.021 and p = 0.001 respectively). Change in lumbar spine BMD was prominent in premenopausal women with advanced clinical stage (IIB, IIIA, IIIB): 0.066 g/cm2 (p = 0.003). CONCLUSION: The antineoplastic breast cancer treatment with chemotherapy had a negative impact on BMD, in premenopausal women overall, although a differential effect was found according to clinical stage and calcium supplementation status.
Assuntos
Antineoplásicos/efeitos adversos , Densidade Óssea , Doenças Ósseas/etiologia , Doenças Ósseas/patologia , Neoplasias da Mama/complicações , Adulto , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Feminino , Humanos , Menopausa , México , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fatores de RiscoRESUMO
The eleven-nineteen lysine-rich leukemia protein (ELL) is a key regulator of RNA polymerase II mediated transcription. ELL facilitates RNA polymerase II transcription pause site entry and release by dynamically interacting with p300 and the positive transcription elongation factor b (P-TEFb). In this study, we investigated the role of ELL during the HTLV-1 Tax oncogene induced transactivation. We show that ectopic expression of Tax enhances ELL incorporation into p300 and P-TEFb containing transcriptional complexes and the subsequent recruitment of these complexes to target genes in vivo. Depletion of ELL abrogates Tax induced transactivation of the immediate early genes Fos, Egr2 and NF-kB, suggesting that ELL is an essential cellular cofactor of the Tax oncogene. Thus, our study identifies a novel mechanism of ELL-dependent transactivation of immediate early genes by Tax and provides the rational for further defining the genome-wide targets of Tax and ELL.
Assuntos
Proteína p300 Associada a E1A/genética , Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Fator B de Elongação Transcricional Positiva/genética , Ativação Transcricional , Fatores de Elongação da Transcrição/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteína p300 Associada a E1A/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Regulação da Expressão Gênica , Produtos do Gene tax/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , NF-kappa B/genética , NF-kappa B/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/metabolismoRESUMO
INTRODUCTION: Population aging is a global phenomenon linked to increased life expectancy. In Argentina, it is expected that by 2025 those over 60 will represent 17.3% of the population, while by 2050 it will rise to 25.3%. Among the pathologies associated with aging, cognitive impairment and dementias represent an important problem for public health and demand effective instruments for their early detection. OBJECTIVE: Obtain normative data for the Montreal Cognitive Assessment (MoCA) in Argentine adults and seniors in the Rosario Metropolitan Area. SUBJECTS AND METHODS: The MoCA-Spanish version was administered according to the instructions published in the original version. An ad hoc survey was also administered to collect sociodemographic information and medical history. The influence of some sociodemographic variables on performance was analyzed. 225 adult residents of the Rosario Metropolitan Area participated in the final sample (age: M = 66.1, standard deviation = 8.7). RESULTS: Educational level predicted 13% of the variance of the total MoCA score, -F (3, 221) = 12.11; p < 0.01-. Other variables considered, such as age and sex, were not significant for predicting the score. CONCLUSION: The normative data obtained suggest a cut-off point of 18 for people with primary education and of 22 for people with secondary or higher education. It should be noted that they are below those indicated in the pre-existing regulatory data. The importance of using norms adjusted to the sociocultural context is highlighted.
TITLE: Montreal Cognitive Assessment (MoCA): normas para la población del área metropolitana de Rosario, Argentina.Introducción. El envejecimiento poblacional es un fenómeno de alcance global ligado al incremento de la expectativa de vida. En Argentina, se prevé que para 2025 los mayores de 60 años representarán el 17,3% de la población, mientras que para 2050 serán el 25,3%. Entre las patologías asociadas al envejecimiento, el deterioro cognitivo y las demencias representan un importante problema para la salud pública y demandan instrumentos eficaces para su detección temprana. Objetivo. Obtener datos normativos para el Montreal Cognitive Assessment (MoCA) en adultos y mayores argentinos del área metropolitana de Rosario. Sujetos y métodos. Se administró el MoCA-versión en español, según las instrucciones publicadas en la versión original. Se administró, además, una encuesta ad hoc para el relevamiento de información sociodemográfica e historial médico. Se analizó la influencia de algunas variables sociodemográficas sobre el desempeño. Participaron en la muestra definitiva 225 adultos residentes del área metropolitana de Rosario (edad: media = 66,1; desviación estándar = 8,7). Resultados. El nivel educativo predijo el 13% de la varianza de la puntuación total del MoCA F (3, 221) = 12,11; p menor de 0,01. Otras variables consideradas, como la edad y el sexo, no resultaron significativas para la predicción de la puntuación. Conclusión. Los datos normativos obtenidos sugieren un punto de corte de 18 para personas con estudios primarios y de 22 para personas con estudios secundarios o superiores. Cabe destacar que éstos se encuentran por debajo de los indicados en los datos normativos preexistentes. Se resalta la importancia de utilizar normas ajustadas al contexto sociocultural.
Assuntos
Cognição , Disfunção Cognitiva , Adulto , Argentina , Disfunção Cognitiva/diagnóstico , Humanos , Testes de Estado Mental e Demência , Testes NeuropsicológicosRESUMO
Interest in the structure, function, and evolutionary relations of circulating and intracellular globins dates back more than 60 years to the first determination of the three-dimensional structure of these proteins. Non-erythrocytic globins have been implicated in circulatory control through reactions that couple nitric oxide (NO) signaling with cellular oxygen availability and redox status. Small artery endothelial cells (ECs) express free α-globin, which causes vasoconstriction by degrading NO. This reaction converts reduced (Fe2+) α-globin to the oxidized (Fe3+) form, which is unstable, cytotoxic, and unable to degrade NO. Therefore, (Fe3+) α-globin must be stabilized and recycled to (Fe2+) α-globin to reinitiate the catalytic cycle. The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) binds (Fe3+) α-globin to inhibit its degradation and facilitate its reduction. The mechanisms that reduce (Fe3+) α-globin in ECs are unknown, although endothelial nitric oxide synthase (eNOS) and cytochrome b5 reductase (CyB5R3) with cytochrome b5 type A (CyB5a) can reduce (Fe3+) α-globin in solution. Here, we examine the expression and cellular localization of eNOS, CyB5a, and CyB5R3 in mouse arterial ECs and show that α-globin can be reduced by either of two independent redox systems, CyB5R3/CyB5a and eNOS. Together, our findings provide new insights into the regulation of blood vessel contractility.
RESUMO
Chronic myeloid leukemia (CML) is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the presence of a Philadelphia (Ph) chromosome. Less than 10% of patients present variant Ph chromosomes involving 1 or more additional chromosomes, other than chromosomes 9 and 22, with uncertain prognosis. There are mainly 1- or 2-step mechanisms proposed to explain the genesis of variant Ph chromosomes depending on whether the involved chromosomes are simultaneously broken and rejoined or if a standard t(9;22) occurs first. By combined standard cytogenetic and FISH analysis we detected a novel variant Ph translocation among chromosomes 9, 11 and 22 in a patient with CML without progression to an accelerated phase of the disease after 7 years, with the derivative chromosome 9 also having an acquired pericentric inversion. This novel case illustrates the use of FISH in metaphase to confirm a new rearrangement not previously described in variant Ph formation and that the present karyotype could have originated by a 1-step mechanism with 4 simultaneous breakages without deletion of ABL1.
Assuntos
Inversão Cromossômica/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 9/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Translocação Genética/genética , Idoso , Antineoplásicos/uso terapêutico , Benzamidas , Bandeamento Cromossômico , Dasatinibe , Feminino , Humanos , Hidroxiureia/uso terapêutico , Mesilato de Imatinib , Hibridização in Situ Fluorescente , Interferon gama/uso terapêutico , Cariotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Metáfase , Cromossomo Filadélfia , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Tiazóis/uso terapêuticoRESUMO
Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.
Assuntos
Histonas/metabolismo , Recombinação Homóloga , Proteínas Mutantes/metabolismo , Schizosaccharomyces/metabolismo , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Histonas/genética , Proteínas Mutantes/genética , Schizosaccharomyces/genéticaRESUMO
A previous report demonstrated that one site in a nucleosome assembled onto a synthetic positioning sequence known as Fragment 67 is hypersensitive to permanganate. The site is required for positioning activity and is located 1.5 turns from the dyad, which is a region of high DNA curvature in the nucleosome. Here, the permanganate sensitivity of the nucleosome positioning Fragment 601 was examined in order to expand the dataset of nucleosome sequences containing KMnO(4) hypersensitive sites. The hyperreactive T residue in the six sites detected as well as the one in Fragment 67 and three in the 5 S rDNA positioning sequence were contained within a TA step. Seven of the ten sequences were of the form CTAGPuG or the related sequence TTAAPu. These motifs were also found in the binding sites of several transcriptional regulatory proteins that kink DNA. In order to assess the significance of these sites, the 10 bp positioning determinant in Fragment 67 was removed and replaced by the nine sequences from the 5 S rDNA and Fragment 601. The results demonstrated that these derivative fragments promoted high nucleosome stability and positioning as compared to a control sequence that contained an AT step in place of the TA step. The importance of the TA step was further tested by making single base-pair substitutions in Fragment 67 and the results revealed that stability and positioning activity followed the order: TA>TG>TT>/=TC approximately GG approximately GA approximately AT. Sequences flanking the TA step were also shown to be critical for nucleosome stability and positioning. Nucleosome positioning was restored to near wild-type levels with (CTG)(3), which can form slipped stranded structures and with one base bulges that kink DNA. The results of this study suggest that local DNA structures are important for positioning and that single base-pair changes at these sites could have profound effects on those genomic functions that depend on ordered nucleosomes.
Assuntos
Nucleossomos/genética , Nucleossomos/metabolismo , Adenina , Animais , Sequência de Bases , Galinhas , DNA/química , DNA/genética , Lytechinus , Compostos de Manganês/farmacologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Óxidos/farmacologia , Sequências Repetitivas de Ácido Nucleico , TiminaRESUMO
Introducción. El envejecimiento poblacional es un fenómeno de alcance global ligado al incremento de la expectativa de vida. En Argentina, se prevé que para 2025 los mayores de 60 años representarán el 17,3% de la población, mientras que para 2050 serán el 25,3%. Entre las patologías asociadas al envejecimiento, el deterioro cognitivo y las demencias representan un importante problema para la salud pública y demandan instrumentos eficaces para su detección temprana. Objetivo. Obtener datos normativos para el Montreal Cognitive Assessment (MoCA) en adultos y mayores argentinos del área metropolitana de Rosario. Sujetos y métodos. Se administró el MoCA-versión en español, según las instrucciones publicadas en la versión original. Se administró, además, una encuesta ad hoc para el relevamiento de información sociodemográfica e historial médico. Se analizó la influencia de algunas variables sociodemográficas sobre el desempeño. Participaron en la muestra definitiva 225 adultos residentes del área metropolitana de Rosario (edad: media = 66,1; desviación estándar = 8,7). Resultados. El nivel educativo predijo el 13% de la varianza de la puntuación total del MoCA F (3, 221) = 12,11; p < 0,01. Otras variables consideradas, como la edad y el sexo, no resultaron significativas para la predicción de la puntuación. Conclusión. Los datos normativos obtenidos sugieren un punto de corte de 18 para personas con estudios primarios y de 22 para personas con estudios secundarios o superiores. Cabe destacar que éstos se encuentran por debajo de los indicados en los datos normativos preexistentes. Se resalta la importancia de utilizar normas ajustadas al contexto sociocultural.(AU)
Introduction. Population aging is a global phenomenon linked to increased life expectancy. In Argentina, it is expected that by 2025 those over 60 will represent 17.3% of the population, while by 2050 it will rise to 25.3%. Among the pathologies associated with aging, cognitive impairment and dementias represent an important problem for public health and demand effective instruments for their early detection. Objective. Obtain normative data for the Montreal Cognitive Assessment (MoCA) in Argentine adults and seniors in the Rosario Metropolitan Area. Subjects and methods. The MoCA-Spanish version was administered according to the instructions published in the original version. An ad hoc survey was also administered to collect sociodemographic information and medical history. The influence of some sociodemographic variables on performance was analyzed. 225 adult residents of the Rosario Metropolitan Area participated in the final sample (age: M = 66.1, standard deviation = 8.7). Results. Educational level predicted 13% of the variance of the total MoCA score, F (3, 221) = 12.11; p < 0.01. Other variables considered, such as age and sex, were not significant for predicting the score. Conclusion. The normative data obtained suggest a cut-off point of 18 for people with primary education and of 22 for people with secondary or higher education. It should be noted that they are below those indicated in the pre-existing regulatory data. The importance of using norms adjusted to the sociocultural context is highlighted.(AU)
Assuntos
Humanos , Pessoa de Meia-Idade , Idoso , Cognição , Disfunção Cognitiva , Neuropsicologia , Envelhecimento/patologia , Dinâmica PopulacionalRESUMO
Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma.
Assuntos
Replicação do DNA , Instabilidade Genômica , Histonas/metabolismo , Recombinação Homóloga , Proteínas Mutantes/metabolismo , Schizosaccharomyces/metabolismo , Reparo do DNA , Histonas/genética , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Schizosaccharomyces/genéticaAssuntos
Antivirais/uso terapêutico , Hepacivirus/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Talassemia/virologia , Adulto , Feminino , Humanos , Interferon alfa-2 , Masculino , Proteínas Recombinantes/uso terapêutico , Resultado do TratamentoRESUMO
UNLABELLED: Recombinant human thyrotropin (rhTSH) has been introduced recently in follow up of differentiated thyroid cancer (DTC) patients, as an alternative of thyroid hormone withdrawal. The aim of this retrospective study is to compare recombinant human thyrotropin versus endogenous stimulation. MATERIAL AND METHODS: Thirty-three patients with DTC with previous thyroidectomy and thyroid ablation were selected. All patients underwent whole-body radioiodine scanning and third day serum thyroglobulin (TG) measurement by two techniques, the first one after conventional thyroid hormone withdrawal (TSHe, TGe), and the second one after rhTSH stimulation (TSHr, TGr). Measurement of TG was performed on the third day due to the infrastructure. We only included patients with stable disease, without therapeutic interventions between two consecutive controls in an interval inferior to one year. Two qualitative categories were defined for TG (positive TG > 2 ng/ml or negative TG<2 ng/ml) and whole-body radioiodine scan (positive or negative). RESULTS: TSHe: 62.9 +/- 55.48; TSHr: 113.16 +/- 50.6; (p: ns); TGe: 62.5 +/- 115.7; TGr: 54.6 +/- 111.1; (p: 0.044). Quantitative data analysis showed significant differences between two techniques. Qualitative data analysis showed no significant differences in clinical setting based in TG and radioiodine scan. CONCLUSIONS: Administration of rhTSH produces a significantly higher increase of TSH than thyroid hormone withdrawal and lower increase in TG levels. There were no significant differences in the stage of disease (TG and whole-body radioiodine scan).