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1.
Acta Pharm ; 73(2): 281-291, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37307375

RESUMO

Methoxamine (Mox) is a well-known α1-adrenoceptor agonist, clinically used as a longer-acting analogue of epinephrine. 1R,2S-Mox (NRL001) has been also undergoing clinical testing to increase the canal resting pressure in patients with bowel incontinence. Here we show, that Mox hydrochloride acts as an inhibitor of base excision repair (BER). The effect is mediated by the inhibition of apurinic/apyrimidinic endonuclease APE1. We link this observation to our previous report showing the biologically relevant effect of Mox on BER - prevention of converting oxidative DNA base damage to double-stranded breaks. We demonstrate that its effect is weaker, but still significant when compared to a known BER inhibitor methoxyamine (MX). We further determined Mox's relative IC 50 at 19 mmol L-1, demonstrating a significant effect of Mox on APE1 activity in clinically relevant concentrations.


Assuntos
Reparo do DNA , Epinefrina , Humanos , Metoxamina , Receptores Adrenérgicos , Endonucleases
2.
BMC Genomics ; 21(1): 677, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998693

RESUMO

BACKGROUND: The mammalian Major Histocompatibility Complex (MHC) is a genetic region containing highly polymorphic genes with immunological functions. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. The MHC class II sub-region contains genes expressed in antigen presenting cells. The antigen binding site is encoded by the second exon of genes encoding antigen presenting molecules. The exon 2 sequences of these MHC genes have evolved under the selective pressure of pathogens. Interspecific differences can be observed in the class II sub-region. The family Equidae includes a variety of domesticated, and free-ranging species inhabiting a range of habitats exposed to different pathogens and represents a model for studying this important part of the immunogenome. While equine MHC class II DRA and DQA loci have received attention, the genetic diversity and effects of selection on DRB and DQB loci have been largely overlooked. This study aimed to provide the first in-depth analysis of the MHC class II DRB and DQB loci in the Equidae family. RESULTS: Three DRB and two DQB genes were identified in the genomes of all equids. The genes DRB2, DRB3 and DQB3 showed high sequence conservation, while polymorphisms were more frequent at DRB1 and DQB1 across all species analyzed. DQB2 was not found in the genome of the Asiatic asses Equus hemionus kulan and E. h. onager. The bioinformatic analysis of non-zero-coverage-bases of DRB and DQB genes in 14 equine individual genomes revealed differences among individual genes. Evidence for recombination was found for DRB1, DRB2, DQB1 and DQB2 genes. Trans-species allele sharing was identified in all genes except DRB1. Site-specific selection analysis predicted genes evolving under positive selection both at DRB and DQB loci. No selected amino acid sites were identified in DQB3. CONCLUSIONS: The organization of the MHC class II sub-region of equids is similar across all species of the family. Genomic sequences, along with phylogenetic trees suggesting effects of selection as well as trans-species polymorphism support the contention that pathogen-driven positive selection has shaped the MHC class II DRB/DQB sub-regions in the Equidae.


Assuntos
Equidae/genética , Evolução Molecular , Complexo Principal de Histocompatibilidade/genética , Polimorfismo Genético , Seleção Genética , Animais , Equidae/classificação , Especiação Genética , Filogenia , Recombinação Genética
3.
Orphanet J Rare Dis ; 15(1): 65, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32138751

RESUMO

We describe the association of Becker muscular dystrophy (BMD) derived heart failure with the impairment of tissue homeostasis and remodeling capabilities of the affected heart tissue. We report that BMD heart failure is associated with a significantly decreased number of cardiovascular progenitor cells, reduced cardiac fibroblast migration, and ex vivo survival. BACKGROUND: Becker muscular dystrophy belongs to a class of genetically inherited dystrophin deficiencies. It affects male patients and results in progressive skeletal muscle degeneration and dilated cardiomyopathy leading to heart failure. It is a relatively mild form of dystrophin deficiency, which allows patients to be on a heart transplant list. In this unique situation, the explanted heart is a rare opportunity to study the degenerative process of dystrophin-deficient cardiac tissue. Heart tissue was excised, dissociated, and analyzed. The fractional content of c-kit+/CD45- cardiovascular progenitor cells (CVPCs) and cardiac fibroblast migration were compared to control samples of atrial tissue. Control tissue was obtained from the hearts of healthy organ donor's during heart transplantation procedures. RESULTS: We report significantly decreased CVPCs (c-kit+/CD45-) throughout the heart tissue of a BMD patient, and reduced numbers of phase-bright cells presenting c-kit positivity in the dystrophin-deficient cultured explants. In addition, ex vivo CVPCs survival and cardiac fibroblasts migration were significantly reduced, suggesting reduced homeostatic support and irreversible tissue remodeling. CONCLUSIONS: Our findings associate genetically derived heart failure in a dystrophin-deficient patient with decreased c-kit+/CD45- CVPCs and their resilience, possibly hinting at a lack of cardioprotective capability and/or reduced homeostatic support. This also correlates with reduced plasticity of the explanted cardiac tissue, related to the process of irreversible remodeling in the BMD patient's heart.


Assuntos
Cardiomiopatia Dilatada , Distrofia Muscular de Duchenne , Distrofina , Humanos , Masculino , Miocárdio , Células-Tronco
4.
FASEB J ; 33(6): 6778-6788, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30807703

RESUMO

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA/fisiologia , Instabilidade Genômica , Células-Tronco Embrionárias Humanas/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Células Cultivadas , Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Ligase Dependente de ATP/genética , Reparo do DNA/efeitos da radiação , Recombinação Homóloga , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética
5.
Cells ; 8(1)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650618

RESUMO

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.


Assuntos
Distrofina/deficiência , Instabilidade Genômica , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Muscular de Duchenne/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Linhagem Celular , Distrofina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
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