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1.
Clin Exp Dermatol ; 49(6): 547-555, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38308656

RESUMO

BACKGROUND: Lipoid proteinosis (LP), also known as Urbach-Wiethe disease, is a rare autosomal recessive genodermatosis, caused by mutations in the ECM1 gene. This results in the deposition of periodic acid-Schiff (PAS)-positive, hyaline-like material on the skin, mucosae and internal organs. OBJECTIVES: To present a case report of LP and a systematic review to synthesize the scientific literature on the management of this uncommon and frequently missed diagnosis. METHODS: We present a case report of a 48-year-old man with LP who exhibited significant improvement after oral acitretin therapy. To address the lack of large case-control studies on LP treatment, we performed a systematic review of the literature following the PRISMA 2020 criteria. The search was conducted in PubMed, Web of Science, Cochrane and Scopus databases from inception until June 2023. To assess the methodological quality of case reports and case series, we used the Joanna Briggs Collaboration critical appraisal tool. RESULTS: We included 25 studies that met eligibility criteria. Data from 44 patients with a histopathologically confirmed diagnosis were analysed. Treatment ranged from systemic therapies (acitretin, etretinate, dimethyl sulfoxide, corticosteroids, penicillamine) to surgical or laser procedures. Regarding methodological quality, the main discrepancies arose in the reporting of participant characteristics and treatment interventions. CONCLUSIONS: Low-dose oral acitretin could have potential in managing LP, exhibiting fewer side-effects compared with other therapeutic agents. Further research is needed to establish more comprehensive and evidence-based treatment guidelines.


Assuntos
Acitretina , Proteinose Lipoide de Urbach e Wiethe , Humanos , Proteinose Lipoide de Urbach e Wiethe/genética , Proteinose Lipoide de Urbach e Wiethe/patologia , Proteinose Lipoide de Urbach e Wiethe/tratamento farmacológico , Proteinose Lipoide de Urbach e Wiethe/diagnóstico , Masculino , Acitretina/uso terapêutico , Pessoa de Meia-Idade , Ceratolíticos/uso terapêutico , Resultado do Tratamento
3.
Mol Cell Proteomics ; 14(7): 1831-45, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25903580

RESUMO

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.


Assuntos
Conexina 43/metabolismo , Osteoartrite/metabolismo , Mapeamento de Interação de Proteínas , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/patologia , Núcleo Celular/metabolismo , Condrócitos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Espectrometria de Massas , Pessoa de Meia-Idade , Osteoartrite/patologia , Ligação Proteica , Transporte Proteico , Vimentina/metabolismo
4.
Ann Rheum Dis ; 74(1): 275-84, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24225059

RESUMO

OBJECTIVE: This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. METHODS: Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. RESULTS: Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and ß-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. CONCLUSIONS: This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.


Assuntos
Cartilagem Articular/metabolismo , Comunicação Celular , Condrócitos/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Aminoácidos Essenciais/metabolismo , Animais , Cartilagem Articular/ultraestrutura , Condrócitos/ultraestrutura , Conexinas/ultraestrutura , Junções Comunicantes/ultraestrutura , Glucose/metabolismo , Homeostase , Humanos , Imuno-Histoquímica , Imunoprecipitação , Articulação do Joelho , Microscopia Eletrônica de Varredura , Suínos
5.
Am J Pathol ; 182(4): 1337-46, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23416160

RESUMO

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 µm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas.


Assuntos
Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Osteoartrite/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Núcleo Celular/metabolismo , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Junções Comunicantes/genética , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo
7.
Fam Cancer ; 23(3): 233-246, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38780705

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the Western world. The number of diagnosed cases and the mortality rate are almost equal as the majority of patients present with advanced disease at diagnosis. Between 4 and 10% of pancreatic cancer cases have an apparent hereditary background, known as hereditary pancreatic cancer (HPC) and familial pancreatic cancer (FPC), when the genetic basis is unknown. Surveillance of high-risk individuals (HRI) from these families by imaging aims to detect PDAC at an early stage to improve prognosis. However, the genetic basis is unknown in the majority of HRIs, with only around 10-13% of families carrying known pathogenic germline mutations. The aim of this study was to assess an individual's genetic cancer risk based on sex and personal and family history of cancer. The Best Linear Unbiased Prediction (BLUP) methodology was used to estimate an individual's predicted risk of developing cancer during their lifetime. The model uses different demographic factors in order to estimate heritability. A reliable estimation of heritability for pancreatic cancer of 0.27 on the liability scale, and 0.07 at the observed data scale as obtained, which is different from zero, indicating a polygenic inheritance pattern of PDAC. BLUP was able to correctly discriminate PDAC cases from healthy individuals and those with other cancer types. Thus, providing an additional tool to assess PDAC risk HRI with an assumed genetic predisposition in the absence of known pathogenic germline mutations.


Assuntos
Carcinoma Ductal Pancreático , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Idoso , Medição de Risco/métodos , Carcinoma
8.
Fam Cancer ; 23(3): 383-392, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38753287

RESUMO

The Spanish Familial Pancreatic Cancer Registry (PANGENFAM) was established in 2009 and aims to characterize the genotype and phenotype of familial pancreatic cancer (FPC). Furthermore, an early detection screening program for pancreatic ductal adenocarcinoma (PDAC) is provided to healthy high-risk individuals from FPC and hereditary pancreatic cancer families (first-degree relatives). This article describes our experience over the last 10 years in high-risk screening. Hereditary and familial pancreatic cancer families were identified through the oncology and gastroenterology units. High-risk individuals underwent annual screening with endoscopic ultrasound (EUS) and magnetic resonance (MRI) from age 40 or 10 years younger than the youngest affected family member. Results: PANGENFAM has enrolled 290 individuals from 143 families, including 52 PDAC cases and 238 high-risk individuals. All high-risk individuals eligible for screening were offered to enter the surveillance program, with 143 currently participating. Pancreatic abnormalities were detected in 94 individuals (median age 53 years (29-83), with common findings including cystic lesions and inhomogeneous parenchyma. Imaging test concordance was 66%. Surgical intervention was performed in 4 high-risk individuals following highly suspicious lesions detected by imaging. PANGENFAM is a valuable resource for science innovation, such as biobanking, with clinical and imaging data available for analysis. For high-risk families, it may offer a potential for early diagnosis. Collaboration with other national and international registries is needed to increase our understanding of the disease biology and to standardize criteria for inclusion and follow-up, optimizing cost-effectiveness and efficacy.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Sistema de Registros , Humanos , Neoplasias Pancreáticas/genética , Pessoa de Meia-Idade , Espanha , Feminino , Masculino , Adulto , Idoso , Seguimentos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/diagnóstico , Idoso de 80 Anos ou mais , Predisposição Genética para Doença , Detecção Precoce de Câncer/métodos , Imageamento por Ressonância Magnética , Endossonografia , Fatores de Risco , Carcinoma
9.
Anticancer Res ; 42(2): 667-674, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35093865

RESUMO

Stereotactic body radiotherapy (SBRT) allows high doses of radiation to be administered in a limited number of fractions. The high doses per session might allow the theoretical radioresistance of renal carcinoma to be overcome. SBRT may be a therapeutic alternative in inoperable patients with localized renal carcinoma. This review studied the available literature on the use of SBRT in inoperable localized renal carcinoma. The review including data from English-language studies was conducted in PubMed and MEDLINE between January 2010 and December 2020. Articles were included with data from patients with renal carcinoma treated with SBRT, their indications, simulation, dose and fractionation, local control, survival and side effects, comparison with other treatments, response assessment and radioimmunotherapy. The articles included were evaluated for content and validation. The immobilization systems were variable between studies. Doses and fractions were variable from 25-26 Gy in single fractions to 21-48 Gy in 3-5 fractions, with local control being around 90% with a low rate of side-effects. We review the state of the art in SBRT for renal cell carcinoma. More research is needed to determine optimal doses and fractionation, and to develop a reliable response assessment tool. The role of radioimmunotherapy in renal carcinoma is being studied.


Assuntos
Carcinoma de Células Renais/radioterapia , Neoplasias Renais/radioterapia , Radiocirurgia , Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células Renais/patologia , Fracionamento da Dose de Radiação , Humanos , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/patologia , Lesões por Radiação/etiologia , Radioimunoterapia , Radiocirurgia/efeitos adversos , Planejamento da Radioterapia Assistida por Computador , Análise de Sobrevida , Resultado do Tratamento
10.
Cancers (Basel) ; 13(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807330

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) presents many challenges in the clinic and there are many areas for improvement in diagnostics and patient management. The five-year survival rate is around 7.2% as the majority of patients present with advanced disease at diagnosis that is treatment resistant. Approximately 10-15% of PDAC cases have a hereditary basis or Familial Pancreatic Cancer (FPC). Here we demonstrate the use of circulating free DNA (cfDNA) in plasma as a prognostic biomarker in PDAC. The levels of cfDNA correlated with disease status, disease stage, and overall survival. Furthermore, we show for the first time via BEAMing that the majority of hereditary or familial PDAC cases (around 84%) are negative for a KRAS somatic mutation. In addition, KRAS mutation negative cases harbor somatic mutations in potentially druggable genes such as KIT, PDGFR, MET, BRAF, and PIK3CA that could be exploited in the clinic. Finally, familial or hereditary cases have a longer overall survival compared to sporadic cases (10.2 vs. 21.7 months, respectively). Currently, all patients are treated the same in the clinic with cytotoxic agents, although here we demonstrate that there are different subtypes of tumors at the genetic level that could pave the way to personalized treatment.

12.
Biomolecules ; 11(1)2020 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-33379193

RESUMO

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damage throughout the cell cycle and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on the early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XRCC4-like factor (XLF), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX) maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.


Assuntos
Proteínas de Ligação a DNA/genética , Células-Tronco Neurais/metabolismo , Células-Tronco/metabolismo , Animais , Apoptose/genética , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Autoantígeno Ku/genética , Camundongos , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Células-Tronco/citologia
13.
Biomolecules ; 10(4)2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32326143

RESUMO

Osteoarthritis (OA) is the most common degenerative joint disease characterized by articular cartilage degradation and joint degeneration. The articular cartilage is mainly formed by chondrocytes and a collagen-proteoglycan extracellular matrix that contains high levels of glycosylated proteins. It was reported that the shift from glycoproteins containing α-2,6-linked sialic acids to those that contain α-2,3 was associated with the onset of common types of arthritis. However, the pathophysiology of α-2,3-sialylation in cartilage has not been yet elucidated. We show that cartilage from osteoarthritic patients expresses high levels of the α-2,3-sialylated transmembrane mucin receptor, known as podoplanin (PDPN). Additionally, the Maackia amurensis seed lectin (MASL), that can be utilized to target PDPN, attenuates the inflammatory response mediated by NF-kB activation in primary chondrocytes and protects human cartilage breakdown ex vivo and in an animal model of arthritis. These findings reveal that specific lectins targeting α-2,3-sialylated receptors on chondrocytes might effectively inhibit cartilage breakdown. We also present a computational 3D molecular model for this interaction. These findings provide mechanistic information on how a specific lectin could be used as a novel therapy to treat degenerative joint diseases such as osteoarthritis.


Assuntos
Osteoartrite/terapia , Receptores de Superfície Celular/metabolismo , Animais , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Feminino , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Lectinas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , NF-kappa B/metabolismo , Osteoartrite/patologia , Ligação Proteica , Isoformas de Proteínas/metabolismo , Transdução de Sinais
14.
DNA Repair (Amst) ; 73: 164-169, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30579708

RESUMO

Non-homologous end joining (NHEJ) is a DNA repair pathway that senses, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. During NHEJ, core Ku70 and Ku80 subunits bind DSBs as a heterodimer and promote further recruitment of accessory factors (e.g., PAXX, Mri, DNA-PKcs, Artemis) and downstream core subunits XRCC4 and DNA ligase 4 (Lig4). Inactivation of Ku70 or Ku80 genes in mice results in immunodeficiency and high levels of genomic instability; deletion of individual Dna-pkcs, Xlf, Paxx or Mri genes results in viable mice with no or modest DNA repair defects. However, combined inactivation of either Xlf and Dna-pkcs, or Xlf and Paxx, or Xlf and Mri, leads to synthetic lethality in mice, which correlates with increased levels of apoptosis in the central nervous system. Here, we demonstrated that inactivation of pro-apoptotic factor Trp53 rescues embryonic lethality of Xlf-/-Paxx-/- and Xlf-/-Dna-pkcs-/- double knockout mice. Moreover, combined inactivation of Paxx and Dna-pkcs results in live-born fertile Paxx-/-Dna-pkcs-/- mice indistinguishable from Dna-pkcs-/- knockout controls.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Inativação Gênica , Mutações Sintéticas Letais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Humanos , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética
15.
Biomolecules ; 9(12)2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795137

RESUMO

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.


Assuntos
Reparo do DNA por Junção de Extremidades/genética , Camundongos Knockout , Animais , Linfócitos B/imunologia , Linhagem Celular , Proliferação de Células , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Switching de Imunoglobulina , Imunoglobulina G/imunologia , Modelos Animais , Células-Tronco , Linfócitos T/imunologia
16.
FEBS Open Bio ; 8(3): 426-434, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29511619

RESUMO

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end-joining (NHEJ) pathway repairs double-strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B-cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx-knockout mice with wild-type (WT) and NHEJ-deficient controls including Ku80- and Dna-pkcs-null and severe combined immunodeficiency mice. Surprisingly, Paxx-deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx-deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx-deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

17.
Biochim Biophys Acta Biomembr ; 1860(12): 2499-2505, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30279151

RESUMO

Cell-to-cell communication between bone, cartilage and the synovial membrane is not fully understood and it is only attributed to the diffusion of substances through the extracellular space or synovial fluid. In this study, we found for the first time that primary bone cells (BCs) including osteocytes, synovial cells (SCs) and chondrocytes (CHs) are able to establish cellular contacts and to couple through gap junction (GJ) channels with connexin43 (Cx43) being dominant. Transwell co-culture and identification by mass spectrometry revealed the exchange of essential amino acids, peptides and proteins including calnexin, calreticulin or CD44 antigen between contacting SCs, BCs and CHs. These results reveal that CHs, SCs and BCs are able to establish intercellular connections and to communicate through GJ channels, which provide a selective signalling route by the direct exchange of potent signalling molecules and metabolites.


Assuntos
Comunicação Celular , Condrócitos/metabolismo , Junções Comunicantes/metabolismo , Osteócitos/metabolismo , Aminoácidos Essenciais/metabolismo , Calnexina/metabolismo , Calreticulina/metabolismo , Células Cultivadas , Técnicas de Cocultura , Conexina 43/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/metabolismo , Transdução de Sinais , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo
18.
FEBS Open Bio ; 8(3): 442-448, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29511621

RESUMO

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining (NHEJ) is the DNA repair process that fixes double-strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination (CSR). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, Artemis, and XLF. Paralog of XRCC4 and XLF (PAXX) is the recently described accessory NHEJ factor that structurally resembles XRCC4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom-generated and commercially available NHEJ-deficient human haploid HAP1 cells, PAXXΔ, XRCC4Δ , and XLFΔ . In our studies, HAP1 PAXXΔ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild-type controls. By contrast, XRCC4Δ and XLFΔ HAP1 cells possessed significant DNA repair defects measured as sensitivity to double-strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR, we generated and characterized Paxx-/- and Aid-/- murine lymphoid CH12F3 cells. CSR to IgA was nearly at wild-type levels in the Paxx-/- cells and completely ablated in the absence of activation-induced cytidine deaminase (AID). In addition, Paxx-/- CH12F3 cells were hypersensitive to zeocin when compared to wild-type controls. We concluded that Paxx-deficient mammalian cells maintain robust NHEJ and CSR.

19.
Oncotarget ; 7(45): 73055-73067, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27682878

RESUMO

Chondrocytes in cartilage and bone cells population express connexin43 (Cx43) and gap junction intercellular communication (GJIC) is essential to synchronize cells for coordinated electrical, mechanical, metabolic and chemical communication in both tissues. Reduced Cx43 connectivity decreases chondrocyte differentiation and defective Cx43 causes skeletal defects. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions. Here we demonstrated that chondrocytes from the CTD-deficient mice, K258stop/Cx43KO and K258stop/K258stop, have reduced GJIC, increased rates of proliferation and reduced expression of collagen type II and proteoglycans. We observed that CTD-truncated mice were significantly smaller in size. Together these results demonstrated that the deletion of the CTD negatively impacts cartilage structure and normal chondrocyte phenotype. These findings suggest that the proteolytic cleavage of the CTD under pathological conditions, such as under the activation of metalloproteinases during tissue injury or inflammation, may account for the deleterious effects of Cx43 in cartilage and bone disorders such as osteoarthritis.


Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Conexina 43/genética , Fenótipo , Domínios e Motivos de Interação entre Proteínas/genética , Animais , Biomarcadores , Cartilagem Articular/patologia , Conexina 43/química , Conexina 43/metabolismo , Matriz Extracelular/metabolismo , Imunofluorescência , Imuno-Histoquímica , Camundongos , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteólise
20.
J Plant Physiol ; 176: 202-9, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25662842

RESUMO

Cotyledon wounding in pepper caused the early generation of hydrogen peroxide both locally (cotyledons) and systemically (upper true leaves). However, 72 h later there is a different wound response between local and systemic organs, as shown by resistance to the pathogenic fungus Botrytis cinerea, that increased locally and decreased systemically. Signaling by ethylene and jasmonic acid was assessed by using two inhibitors: 1-methylcyclopropene (MCP, inhibitor of ethylene receptors) and ibuprofen (inhibitor of jasmonate biosynthesis). MCP did not affect the modulation of resistance levels to Botrytis by wounding, ruling out the involvement of ethylene signaling. Ibuprofen did not inhibit wound-induced resistance at the local level, but inhibited wound-induced systemic susceptibility. Moreover, changes of biochemical and structural defenses in response to wounding were studied. Peroxidase activity and the expression of a peroxidase gene (CAPO1) increased locally as a response to wounding, but no changes were observed systemically. Lignin deposition was induced in wounded cotyledons, but was repressed in systemic leaves of wounded plants, whereas soluble phenolics did not change locally and decreased systemically. The expression of two other genes involved in plant defense (CABPR1 and CASC1) was also differentially regulated locally and systemically, pointing to a generalized increase in plant defenses at the local level and a systemic decrease as a response to wounding. Wound-induced defenses at the local level coincided with resistance to the necrotroph fungus B. cinerea, whereas depleted defenses in systemic leaves of wounded plants correlated to induced susceptibility against this pathogen. It may be that the local response acts as a sink of energy resources to mount a defense against pathogens, whereas in systemic organs the resources for defense are lower.


Assuntos
Botrytis/fisiologia , Capsicum/imunologia , Capsicum/microbiologia , Resistência à Doença/imunologia , Doenças das Plantas/microbiologia , Botrytis/efeitos dos fármacos , Capsicum/efeitos dos fármacos , Capsicum/enzimologia , Quitinases/metabolismo , Cotilédone/efeitos dos fármacos , Cotilédone/metabolismo , Ciclopentanos/metabolismo , Ciclopropanos/farmacologia , Resistência à Doença/efeitos dos fármacos , Suscetibilidade a Doenças , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Ibuprofeno/farmacologia , Lignina/metabolismo , Oxilipinas/metabolismo , Peroxidase/metabolismo , Fenóis/metabolismo , Doenças das Plantas/imunologia , Solubilidade
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