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1.
J Immunol ; 210(11): 1804-1814, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37074207

RESUMO

Somatic hypermutation (SHM) is necessary for Ab diversification and involves error-prone DNA repair of activation-induced cytidine deaminase-induced lesions in germinal center (GC) B cells but can also cause genomic instability. GC B cells express low levels of the DNA repair protein apurinic/apyrimidinic (AP) endonuclease (APE)1 and high levels of its homolog APE2. Reduced SHM in APE2-deficient mice suggests that APE2 promotes SHM, but these GC B cells also exhibit reduced proliferation that could impact mutation frequency. In this study, we test the hypothesis that APE2 promotes and APE1 suppresses SHM. We show how APE1/APE2 expression changes in primary murine spleen B cells during activation, impacting both SHM and class-switch recombination (CSR). High levels of both APE1 and APE2 early after activation promote CSR. However, after 2 d, APE1 levels decrease steadily with each cell division, even with repeated stimulation, whereas APE2 levels increase with each stimulation. When GC-level APE1/APE2 expression was engineered by reducing APE1 genetically (apex1+/-) and overexpressing APE2, bona fide activation-induced cytidine deaminase-dependent VDJH4 intron SHM became detectable in primary B cell cultures. The C terminus of APE2 that interacts with proliferating cell nuclear Ag promotes SHM and CSR, although its ATR-Chk1-interacting Zf-GRF domain is not required. However, APE2 does not increase mutations unless APE1 is reduced. Although APE1 promotes CSR, it suppresses SHM, suggesting that downregulation of APE1 in the GC is required for SHM. Genome-wide expression data compare GC and cultured B cells and new models depict how APE1 and APE2 expression and protein interactions change during B cell activation and affect the balance between accurate and error-prone repair during CSR and SHM.


Assuntos
Linfócitos B , Reparo do DNA , Animais , Camundongos , Linfócitos B/metabolismo , Técnicas de Cultura de Células , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Switching de Imunoglobulina/genética , Mutação , Hipermutação Somática de Imunoglobulina
2.
J Infect Chemother ; 30(5): 423-428, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-37981024

RESUMO

INTRODUCTION: Cefmetazole (CMZ), an antibiotic with limited international distribution, is recommended by the Tokyo Guidelines 2018 (TG18) for non-severe cases of acute cholangitis (AC). However, the risk factors for CMZ-non-susceptible (CMZ-NS) bacteremia in AC remain unclear. Here, we aimed to investigate the risk factors for CMZ-NS bacteremia and evaluate mortality in patients with AC. METHODS: This single-center, retrospective, observational study included all patients diagnosed with definite bacteremic AC, based on TG18, from April 2019 to March 2023. Risk factors for CMZ-NS bacteremia were analyzed by univariate, and age- and sex-adjusted, logistic regression analyses. Mortality was compared by cause of obstruction, CMZ-susceptible/CMZ-NS bacteremia, and initial treatment. RESULTS: In total, 165 patients were enrolled. CMZ-NS bacteremia was diagnosed in 46 (27.9 %) patients. Histories of diabetes mellitus, hepato-biliary-pancreatic cancer, malignant biliary obstruction, and endoscopic sphincterotomy were identified as significant factors associated with the risk of CMZ-NS bacteremia. Thirteen patients died within 30 days of hospital admission. The mortality of patients with AC and malignant biliary obstruction was statistically higher than that of patients with bile duct stones. No patients with AC and bile duct stones died in the group with CMZ-NS bacteremia and inappropriate initial antibiotics. CONCLUSIONS: In AC, a history of diabetes mellitus, hepato-biliary-pancreatic cancer, malignant biliary obstruction, and endoscopic sphincterotomy are associated with an increased risk of CMZ-NS bacteremia. Therefore, the choice of empiric therapy for AC should be based on the etiology and patient background, rather than on the severity.


Assuntos
Colangite , Colestase , Diabetes Mellitus , Neoplasias Pancreáticas , Humanos , Antibacterianos/uso terapêutico , Cefmetazol , Colangite/complicações , Colangite/tratamento farmacológico , Neoplasias Pancreáticas/complicações , Estudos Retrospectivos , Fatores de Risco , Masculino , Feminino
3.
Biol Pharm Bull ; 45(9): 1340-1346, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36047203

RESUMO

This study aimed to clarify the details of outpatient oral antimicrobial use (AMU) at a Japanese community hospital and investigate the influence of the current inpatient-based antimicrobial stewardship (AS) on outpatients. A repeated cross-sectional study was conducted in Komaki City Hospital. Data on patients, physicians, and oral antibiotics were collected in October 2013, 2016, and 2019, and appropriateness of treatment and surgical antimicrobial prophylaxis (SAP) was evaluated. The percentage of patients receiving oral antibiotics increased significantly from 4.7% in 2013 (345/7338) to 5.9% in 2019 (365/6146), and the overall number of antimicrobial prescriptions per 1000 outpatients increased from 51.8 in 2013 to 68.0 in 2019. Prescriptions for third-generation cephalosporins per 1000 outpatients decreased (from 21.4 to 6.3), whereas the number of prescriptions for penicillin (from 3.8 to 15.3), fluoroquinolones (from 7.0 to 13.2), and co-trimoxazole (from 5.0 to 15.8) increased from 2013 to 2019. The appropriate AMU for overall infections significantly increased (from 68.4% in 2013 to 83.7% in 2019). The choice and duration of AMU significantly improved for SAP. However, even in 2019, only 29.3% of patients received antibiotics before surgery. The improved selection of antibiotics on outpatient prescription may be due to the influence of AS-which is focused on inpatients-while prescriptions for fluoroquinolones and prophylactics also increased. The challenges of antimicrobial administration after surgeries were also highlighted.


Assuntos
Anti-Infecciosos , Gestão de Antimicrobianos , Antibacterianos/uso terapêutico , Anti-Infecciosos/uso terapêutico , Estudos Transversais , Prescrições de Medicamentos , Fluoroquinolonas , Hospitais Comunitários , Humanos , Pacientes Internados , Japão , Pacientes Ambulatoriais
4.
Ann Neurol ; 84(2): 208-224, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30014510

RESUMO

OBJECTIVE: To identify novel autoantibodies for neuropathic pain (NeP). METHODS: We screened autoantibodies that selectively bind to mouse unmyelinated C-fiber type dorsal root ganglion (DRG) neurons using tissue-based indirect immunofluorescence assays (IFA) with sera from 110 NeP patients with various inflammatory and allergic neurologic diseases or other neuropathies, and 50 controls without NeP including 20 healthy subjects and 30 patients with neurodegenerative diseases or systemic inflammatory diseases. IgG purified from IFA-positive patients' sera was subjected to Western blotting (WB) and immunoprecipitation (IP) using mouse DRG lysates. Immunoprecipitates were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify target autoantigens. RESULTS: Antiunmyelinated C-fiber type DRG neuron antibodies were more frequent in patients with NeP than non-NeP subjects (10% vs 0%; p < 0.05). These autoantibodies were all from the IgG2 subclass and colocalized mostly with isolectin B4- and P2X3-positive pain-conducting small neurons but not with S100ß-positive myelinated neurons. WB revealed a common immunoreactive band (approximately 220kDa). IP and LC-MS/MS studies identified plexin D1 as a target autoantigen. Immunoadsorption tests with recombinant human plexin D1 in IFA revealed that all 11 anti-small DRG neuron antibody-positive patients had anti-plexin D1 antibodies. Application of anti-plexin D1 antibody-positive patient sera to cultured DRG neurons increased membrane permeability, leading to cellular swelling. NeP patients with anti-plexin D1 antibodies commonly developed burning pain and current perception threshold abnormalities for C-fibers. Main comorbidities were atopy and collagen-vascular disease. Immunotherapies ameliorated NeP in 7 treated cases. INTERPRETATION: Anti-plexin D1 antibodies are a novel biomarker for immunotherapy-responsive NeP. Ann Neurol 2018;84:208-224.


Assuntos
Autoanticorpos/sangue , Moléculas de Adesão Celular Neuronais/sangue , Neuralgia/sangue , Neuralgia/diagnóstico , Adulto , Idoso , Animais , Biomarcadores/sangue , Células Cultivadas , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Células HeLa , Humanos , Imunoterapia/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neuralgia/terapia , Estudos Retrospectivos , Adulto Jovem
5.
Genes Cells ; 21(10): 1030-1048, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27545963

RESUMO

Genomewide association studies have shown that a nonsynonymous single nucleotide polymorphism in PRKCH is associated with cerebral infarction and atherosclerosis-related complications. We examined the role of PKCη in lipid metabolism and atherosclerosis using apolipoprotein E-deficient (Apoe-/- ) mice. PKCη expression was augmented in the aortas of mice with atherosclerosis and exclusively detected in MOMA2-positive macrophages within atherosclerotic lesions. Prkch+/+ Apoe-/- and Prkch-/- Apoe-/- mice were fed a high-fat diet (HFD), and the dyslipidemia observed in Prkch+/+ Apoe-/- mice was improved in Prkch-/- Apoe-/- mice, with a particular reduction in serum LDL cholesterol and phospholipids. Liver steatosis, which developed in Prkch+/+ Apoe-/- mice, was improved in Prkch-/- Apoe-/- mice, but glucose tolerance, adipose tissue and body weight, and blood pressure were unchanged. Consistent with improvements in LDL cholesterol, atherosclerotic lesions were decreased in HFD-fed Prkch-/- Apoe-/- mice. Immunoreactivity against 3-nitrotyrosine in atherosclerotic lesions was dramatically decreased in Prkch-/- Apoe-/- mice, accompanied by decreased necrosis and apoptosis in the lesions. ARG2 mRNA and protein levels were significantly increased in Prkch-/- Apoe-/- macrophages. These data show that PKCη deficiency improves dyslipidemia and reduces susceptibility to atherosclerosis in Apoe-/- mice, showing that PKCη plays a role in atherosclerosis development.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Metabolismo dos Lipídeos , Proteína Quinase C/deficiência , Animais , Aorta/metabolismo , Apoptose , Aterosclerose/patologia , Dieta Hiperlipídica , Suscetibilidade a Doenças , Dislipidemias/metabolismo , Fígado Gorduroso/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Estresse Oxidativo
6.
Proc Natl Acad Sci U S A ; 111(25): 9217-22, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24927551

RESUMO

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase ß. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.


Assuntos
Linfócitos B/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Endonucleases/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Centro Germinativo/metabolismo , Mutação , Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Linfócitos B/citologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Centro Germinativo/citologia , Camundongos , Camundongos Knockout , Enzimas Multifuncionais , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo
7.
J Immunol ; 193(2): 931-9, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24935922

RESUMO

Activation-induced cytidine deaminase (AID) initiates a process generating DNA mutations and breaks in germinal center (GC) B cells that are necessary for somatic hypermutation and class-switch recombination. GC B cells can "tolerate" DNA damage while rapidly proliferating because of partial suppression of the DNA damage response by BCL6. In this study, we develop a model to study the response of mouse GC B cells to endogenous DNA damage. We show that the base excision repair protein apurinic/apyrimidinic endonuclease (APE) 2 protects activated B cells from oxidative damage in vitro. APE2-deficient mice have smaller GCs and reduced Ab responses compared with wild-type mice. DNA double-strand breaks are increased in the rapidly dividing GC centroblasts of APE2-deficient mice, which activate a p53-independent cell cycle checkpoint and a p53-dependent apoptotic response. Proliferative and/or oxidative damage and AID-dependent damage are additive stresses that correlate inversely with GC size in wild-type, AID-, and APE2-deficient mice. Excessive double-strand breaks lead to decreased expression of BCL6, which would enable DNA repair pathways but limit GC cell numbers. These results describe a nonredundant role for APE2 in the protection of GC cells from AID-independent damage, and although GC cells uniquely tolerate DNA damage, we find that the DNA damage response can still regulate GC size through pathways that involve p53 and BCL6.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/imunologia , Dano ao DNA , Endonucleases/imunologia , Centro Germinativo/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Linfócitos B/metabolismo , Ciclo Celular/genética , Ciclo Celular/imunologia , Proliferação de Células , Células Cultivadas , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Endonucleases/deficiência , Endonucleases/genética , Citometria de Fluxo , Centro Germinativo/metabolismo , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Enzimas Multifuncionais , Estresse Oxidativo/imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
8.
Glia ; 62(8): 1284-98, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24771617

RESUMO

The Fosb gene encodes subunits of the activator protein-1 transcription factor complex. Two mature mRNAs, Fosb and ΔFosb, encoding full-length FOSB and ΔFOSB proteins respectively, are formed by alternative splicing of Fosb mRNA. Fosb products are expressed in several brain regions. Moreover, Fosb-null mice exhibit depressive-like behaviors and adult-onset spontaneous epilepsy, demonstrating important roles in neurological and psychiatric disorders. Study of Fosb products has focused almost exclusively on neurons; their function in glial cells remains to be explored. In this study, we found that microglia express equivalent levels of Fosb and ΔFosb mRNAs to hippocampal neurons and, using microarray analysis, we identified six microglial genes whose expression is dependent on Fosb products. Of these genes, we focused on C5ar1 and C5ar2, which encode receptors for complement C5a. In isolated Fosb-null microglia, chemotactic responsiveness toward the truncated form of C5a was significantly lower than that in wild-type cells. Fosb-null mice were significantly resistant to kainate-induced seizures compared with wild-type mice. C5ar1 mRNA levels and C5aR1 immunoreactivity were increased in wild-type hippocampus 24 hours after kainate administration; however, such induction was significantly reduced in Fosb-null hippocampus. Furthermore, microglial activation after kainate administration was significantly diminished in Fosb-null hippocampus, as shown by significant reductions in CD68 immunoreactivity, morphological change and reduced levels of Il6 and Tnf mRNAs, although no change in the number of Iba-1-positive cells was observed. These findings demonstrate that, under excitotoxicity, Fosb products contribute to a neuroinflammatory response in the hippocampus through regulation of microglial C5ar1 and C5ar2 expression.


Assuntos
Hipocampo/fisiopatologia , Microglia/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Processamento Alternativo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Astrócitos/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Quimiotaxia/fisiologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Interleucina-6/metabolismo , Ácido Caínico/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Convulsões/patologia , Convulsões/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo
9.
J Immunol ; 186(4): 1943-50, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21228350

RESUMO

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.


Assuntos
Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Endonucleases/fisiologia , Fluoruracila/administração & dosagem , Células-Tronco Hematopoéticas/enzimologia , Subpopulações de Linfócitos/enzimologia , Linfopoese/imunologia , Animais , Subpopulações de Linfócitos B/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/imunologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Reparo do DNA/imunologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Endonucleases/deficiência , Endonucleases/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Depleção Linfocítica , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Linfopoese/efeitos dos fármacos , Linfopoese/genética , Camundongos , Camundongos Knockout , Enzimas Multifuncionais , Mielopoese/efeitos dos fármacos , Mielopoese/genética , Mielopoese/imunologia , Proteína Supressora de Tumor p53/fisiologia
10.
EMBO J ; 27(2): 421-32, 2008 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-18188152

RESUMO

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.


Assuntos
Núcleo Celular/genética , Dano ao DNA , DNA Mitocondrial/metabolismo , Transdução de Sinais , 8-Hidroxi-2'-Desoxiguanosina , Animais , Fator de Indução de Apoptose/metabolismo , Western Blotting , Cálcio/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Morte Celular/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Ensaio Cometa , Quebras de DNA de Cadeia Simples , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Humanos , Camundongos , Mutação , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , Transfecção , Vitamina K 3/farmacologia
11.
Nucleic Acids Res ; 38(14): 4834-43, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20385596

RESUMO

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Quebras de DNA de Cadeia Simples , Pirofosfatases/metabolismo , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Sequência de Aminoácidos , Núcleo Celular/química , Proliferação de Células , Técnicas de Silenciamento de Genes , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Nucleotídeos de Inosina/metabolismo , Inosina Trifosfato/metabolismo , Dados de Sequência Molecular , Pirofosfatases/deficiência , Pirofosfatases/genética , Ribonucleotídeos/metabolismo
12.
Nucleic Acids Res ; 38(9): 2891-903, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20081199

RESUMO

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.


Assuntos
Hidrolases Anidrido Ácido/fisiologia , Instabilidade Cromossômica , Inosina Difosfato/metabolismo , Inosina Trifosfato/metabolismo , Pirofosfatases/fisiologia , Hidrolases Anidrido Ácido/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Nucleotídeos de Inosina/metabolismo , Inosina Trifosfato/análogos & derivados , Camundongos , Camundongos Knockout , Fenótipo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Inosina Trifosfatase
13.
Sci Rep ; 11(1): 5819, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758207

RESUMO

8-Oxoguanine (8-oxoG), a major oxidative base lesion, is highly accumulated in Alzheimer's disease (AD) brains during the pathogenic process. MTH1 hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby avoiding 8-oxo-dG incorporation into DNA. 8-OxoG DNA glycosylase-1 (OGG1) excises 8-oxoG paired with cytosine in DNA, thereby minimizing 8-oxoG accumulation in DNA. Levels of MTH1 and OGG1 are significantly reduced in the brains of sporadic AD cases. To understand how 8-oxoG accumulation in the genome is involved in AD pathogenesis, we established an AD mouse model with knockout of Mth1 and Ogg1 genes in a 3xTg-AD background. MTH1 and OGG1 deficiency increased 8-oxoG accumulation in nuclear and, to a lesser extent, mitochondrial genomes, causing microglial activation and neuronal loss with impaired cognitive function at 4-5 months of age. Furthermore, minocycline, which inhibits microglial activation and reduces neuroinflammation, markedly decreased the nuclear accumulation of 8-oxoG in microglia, and inhibited microgliosis and neuronal loss. Gene expression profiling revealed that MTH1 and OGG1 efficiently suppress progression of AD by inducing various protective genes against AD pathogenesis initiated by Aß/Tau accumulation in 3xTg-AD brain. Our findings indicate that efficient suppression of 8-oxoG accumulation in brain genomes is a new approach for prevention and treatment of AD.


Assuntos
Doença de Alzheimer/genética , DNA Glicosilases/genética , Guanina/análogos & derivados , Monoéster Fosfórico Hidrolases/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Progressão da Doença , Perfilação da Expressão Gênica , Guanina/metabolismo , Guanina/toxicidade , Humanos , Camundongos , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Estresse Oxidativo/efeitos dos fármacos
14.
Front Neurol ; 12: 681980, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34421790

RESUMO

Multiple sclerosis (MS), the most prevalent inflammatory disease of the central nervous system (CNS), is characterized by damaged to myelin sheaths and oligodendrocytes. Because MS patients have variable clinical courses and disease severities, it is important to identify biomarkers that predict disease activity and severity. In this study, we assessed the frequencies of serum autoantibodies against mature oligodendrocytes in MS patients using a tissue-based immunofluorescence assay (IFA) to determine whether anti-oligodendrocyte antibodies are associated with the clinical features of MS patients and whether they might be a biomarker to assess CNS tissue damage in MS patients. We assessed the binding of serum autoantibodies to mouse oligodendrocytes expressing Nogo-A, a reliable mature oligodendrocyte marker, by IFA with mouse brain and sera from 147 MS patients, comprising 103 relapsing-remitting MS (RRMS), 22 secondary progressive MS (SPMS), and 22 primary progressive MS (PPMS) patients, 38 neuromyelitis optica spectrum disorder (NMOSD) patients, 23 other inflammatory neurological disorder (OIND) patients, and 39 healthy controls (HCs). Western blotting (WB) was performed using extracted mouse cerebellum proteins and IgG from anti-oligodendrocyte antibody-positive MS patients. Tissue-based IFA showed that anti-oligodendrocyte antibodies were positive in 3/22 (13.6%) PPMS and 1/22 (4.5%) SPMS patients but not in RRMS, NMOSD, and OIND patients or HCs. WB demonstrated the target CNS proteins recognized by serum anti-oligodendrocyte antibodies were approximately 110 kDa and/or 150 kDa. Compared with anti-oligodendrocyte antibody-negative MS patients, MS patients with anti-oligodendrocyte antibodies were significantly older at the time of serum sampling, scored significantly higher on the Expanded Disability Status Scale and the Multiple Sclerosis Severity Score, and had a higher frequency of mental disturbance. Although the clinical significance of anti-oligodendrocyte antibodies is still unclear because of their low frequency, anti-oligodendrocyte autoantibodies are potential biomarkers for monitoring the disease pathology and progression in MS.

15.
Front Cell Dev Biol ; 9: 738502, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34796173

RESUMO

The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3'-5' exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.

16.
Cancer Res ; 81(3): 713-723, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33288657

RESUMO

Cisplatin chemotherapy is standard care for many cancers but is toxic to the kidneys. How this toxicity occurs is uncertain. In this study, we identified apurinic/apyrimidinic endonuclease 2 (APE2) as a critical molecule upregulated in the proximal tubule cells (PTC) following cisplatin-induced nuclear DNA and mitochondrial DNA damage in cisplatin-treated C57B6J mice. The APE2 transgenic mouse phenotype recapitulated the pathophysiological features of C-AKI (acute kidney injury, AKI) in the absence of cisplatin treatment. APE2 pulldown-MS analysis revealed that APE2 binds myosin heavy-Chain 9 (MYH9) protein in mitochondria after cisplatin treatment. Human MYH9-related disorder is caused by mutations in MYH9 that eventually lead to nephritis, macrothrombocytopenia, and deafness, a constellation of symptoms similar to the toxicity profile of cisplatin. Moreover, cisplatin-induced C-AKI was attenuated in APE2-knockout mice. Taken together, these findings suggest that cisplatin promotes AKI development by upregulating APE2, which leads to subsequent MYH9 dysfunction in PTC mitochondria due to an unrelated role of APE2 in DNA damage repair. This postulated mechanism and the availability of an engineered transgenic mouse model based on the mechanism of C-AKI provides an opportunity to identify novel targets for prophylactic treatment of this serious disease. SIGNIFICANCE: These results reveal and highlight an unexpected role of APE2 via its interaction with MYH9 and suggest that APE2 has the potential to prevent acute kidney injury in patients with cisplatin-treated cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/713/F1.large.jpg.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Enzimas Multifuncionais/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Carboplatina/efeitos adversos , Dano ao DNA , DNA Mitocondrial/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/efeitos dos fármacos , Endonucleases/genética , Perda Auditiva Neurossensorial/induzido quimicamente , Humanos , Túbulos Renais Proximais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Enzimas Multifuncionais/efeitos dos fármacos , Enzimas Multifuncionais/genética , Mutação , Cadeias Pesadas de Miosina/genética , Nefrite/induzido quimicamente , Oxaliplatina/efeitos adversos , Fenótipo , Trombocitopenia/induzido quimicamente , Regulação para Cima/efeitos dos fármacos
17.
Int Immunol ; 21(8): 947-55, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19556307

RESUMO

The DNA cleavage step in both the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID). However, the detailed mechanisms of the DNA strand cleavage in SHM and CSR are still largely unknown. Recently, the apurinic/apyrimidinic endonucleases, Apex1 and Apex2, were reported to be involved in the DNA cleavage step of CSR. Here, we examined the role of Apex2 in SHM using Apex2-deficient mice and found that the Apex2 deficiency caused a drastic reduction in the frequency of SHM and the number of mutations per mutated clone without affecting the pattern of base substitution. These results suggest that Apex2 may play a critical role in SHM through its 3'-5' exonuclease activity. Unexpectedly, the efficiency of CSR was not reduced in Apex2-deficient B cells. In addition, Apex1 knockdown in CH12F3-2 B lymphoma cells did not affect the CSR frequency, suggesting that neither Apex1 nor Apex2 plays a major role in CSR.


Assuntos
Endonucleases/fisiologia , Genes de Imunoglobulinas , Switching de Imunoglobulina , Hipermutação Somática de Imunoglobulina/genética , Animais , Linfócitos B/enzimologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Endonucleases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enzimas Multifuncionais , Recombinação Genética
18.
Mutat Res ; 703(1): 51-8, 2010 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-20542142

RESUMO

Accumulation of oxidized bases such as 8-oxoguanine in either nuclear or mitochondrial DNA triggers various cellular dysfunctions including mutagenesis, and programmed cell death or senescence. Recent studies have revealed that oxidized nucleoside triphosphates such as 8-oxo-dGTP in the nucleotide pool are the main source of oxidized bases accumulating in the DNA of cells under oxidative stress. To counteract such deleterious effects of nucleotide pool damage, mammalian cells possess MutT homolog-1 (MTH1) with oxidized purine nucleoside triphosphatase and related enzymes, thus minimizing the accumulation of oxidized bases in cellular DNA. Depletion or increased expression of the MTH1 protein have revealed its significant roles in avoiding programmed cell death or senescence as well as mutagenesis, and accumulating evidences indicate that MTH1 is involved in suppression of degenerative disorders such as neurodegeneration.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Apoptose , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , DNA Mitocondrial/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Degeneração Neural , Neurônios/metabolismo , Estresse Oxidativo/genética , Nucleosídeos de Purina/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Mutat Res ; 703(1): 43-50, 2010 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-20601097

RESUMO

Inosine triphosphate pyrophosphatase (ITPA protein) (EC 3.6.1.19) hydrolyzes deaminated purine nucleoside triphosphates, such as ITP and dITP, to their corresponding purine nucleoside monophosphate and pyrophosphate. In mammals, this enzyme is encoded by the Itpa gene. Using the Itpa gene-disrupted mouse as a model, we have elucidated the biological significance of the ITPA protein and its substrates, ITP and dITP. Itpa(-/-) mice exhibited peri- or post-natal lethality dependent on the genetic background. The heart of the Itpa(-/-) mouse was found to be structurally and functionally abnormal. Significantly higher levels of deoxyinosine and inosine were detected in nuclear DNA and RNA prepared from Itpa(-/-) embryos compared to wild type embryos. In addition, an accumulation of ITP was observed in the erythrocytes of Itpa(-/-) mice. We found that Itpa(-/-) primary mouse embryonic fibroblasts (MEFs), which have no detectable ability to generate IMP from ITP in whole cell extracts, exhibited a prolonged population-doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in their nuclear DNA, in comparison to primary MEFs prepared from wild type embryos. These results revealed that (1) ITP and dITP are spontaneously produced in vivo and (2) accumulation of ITP and dITP is responsible for the harmful effects observed in the Itpa(-/-) mouse. In addition to its effect as the precursor nucleotide for RNA transcription, ITP has the potential to influence the activity of ATP/GTP-binding proteins. The biological significance of ITP and dITP in the nucleotide pool remains to be elucidated.


Assuntos
Nucleotídeos de Purina/metabolismo , Pirofosfatases/metabolismo , Animais , Aberrações Cromossômicas , DNA/química , Desaminação , Cardiopatias Congênitas/genética , Humanos , Camundongos , Camundongos Transgênicos , Pirofosfatases/genética , RNA/química , Transdução de Sinais
20.
Mutat Res ; 703(1): 37-42, 2010 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-20542141

RESUMO

To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.


Assuntos
Proteínas de Transporte/isolamento & purificação , Nucleotídeos/metabolismo , Proteômica/métodos , Pirofosfatases/isolamento & purificação , Animais , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Camundongos , Pirofosfatases/genética , Estudos de Validação como Assunto , Inosina Trifosfatase
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