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1.
Am J Hum Genet ; 108(1): 115-133, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33308444

RESUMO

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.


Assuntos
Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Deficiências do Desenvolvimento/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL
2.
Int J Mol Sci ; 24(9)2023 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-37175696

RESUMO

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Sistemas CRISPR-Cas/genética , Endofenótipos , Diferenciação Celular/genética , Edição de Genes , Mutação , Proteínas Associadas aos Microtúbulos/metabolismo
3.
Clin Genet ; 102(1): 12-21, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35396703

RESUMO

Prompt diagnosis of complex phenotypes is a challenging task in clinical genetics. Whole exome sequencing has proved to be effective in solving such conditions. Here, we report on an unpredictable presentation of Werner Syndrome (WRNS) in a 12-year-old girl carrying a homozygous truncating variant in RECQL2, the gene mutated in WRNS, and a de novo activating missense change in PTPN11, the major Noonan syndrome gene, encoding SHP2, a protein tyrosine phosphatase positively controlling RAS function and MAPK signaling, which have tightly been associated with senescence in primary cells. All the major WRNS clinical criteria were present with an extreme precocious onset and were associated with mild intellectual disability, severe growth retardation and facial dysmorphism. Compared to primary fibroblasts from adult subjects with WRNS, proband's fibroblasts showed a dramatically reduced proliferation rate and competence, and a more accelerated senescence, in line with the anticipated WRNS features occurring in the child. In vitro functional characterization of the SHP2 mutant documented its hyperactive behavior and a significantly enhanced activation of the MAPK pathway. Based on the functional interaction of WRN and MAPK signaling in processes relevant to replicative senescence, these findings disclose a unique phenotype likely resulting from negative genetic interaction.


Assuntos
Síndrome de Noonan , Síndrome de Werner , Criança , Mutação com Ganho de Função , Humanos , Mutação , Síndrome de Noonan/genética , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Síndrome de Werner/genética
4.
Int J Mol Sci ; 22(24)2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34948465

RESUMO

To date, gene therapy has employed viral vectors to deliver therapeutic genes. However, recent progress in molecular and cell biology has revolutionized the field of stem cells and gene therapy. A few years ago, clinical trials started using stem cell replacement therapy, and the induced pluripotent stem cells (iPSCs) technology combined with CRISPR-Cas9 gene editing has launched a new era in gene therapy for the treatment of neurological disorders. Here, we summarize the latest findings in this research field and discuss their clinical applications, emphasizing the relevance of recent studies in the development of innovative stem cell and gene editing therapeutic approaches. Even though tumorigenicity and immunogenicity are existing hurdles, we report how recent progress has tackled them, making engineered stem cell transplantation therapy a realistic option.


Assuntos
Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Doenças do Sistema Nervoso/terapia , Transplante de Células-Tronco/métodos , Sistemas CRISPR-Cas , Ensaios Clínicos como Assunto , Edição de Genes , Humanos , Doenças do Sistema Nervoso/genética
5.
Am J Med Genet A ; 170(10): 2681-93, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27311832

RESUMO

Mutations in CREBBP cause Rubinstein-Taybi syndrome. By using exome sequencing, and by using Sanger in one patient, CREBBP mutations were detected in 11 patients who did not, or only in a very limited manner, resemble Rubinstein-Taybi syndrome. The combined facial signs typical for Rubinstein-Taybi syndrome were absent, none had broad thumbs, and three had only somewhat broad halluces. All had apparent developmental delay (being the reason for molecular analysis); five had short stature and seven had microcephaly. The facial characteristics were variable; main characteristics were short palpebral fissures, telecanthi, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum. Six patients had autistic behavior, and two had self-injurious behavior. Other symptoms were recurrent upper airway infections (n = 5), feeding problems (n = 7) and impaired hearing (n = 7). Major malformations occurred infrequently. All patients had a de novo missense mutation in the last part of exon 30 or beginning of exon 31 of CREBBP, between base pairs 5,128 and 5,614 (codons 1,710 and 1,872). No missense or truncating mutations in this region have been described to be associated with the classical Rubinstein-Taybi syndrome phenotype. No functional studies have (yet) been performed, but we hypothesize that the mutations disturb protein-protein interactions by altering zinc finger function. We conclude that patients with missense mutations in this specific CREBBP region show a phenotype that differs substantially from that in patients with Rubinstein-Taybi syndrome, and may prove to constitute one (or more) separate entities. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteína de Ligação a CREB/genética , Estudos de Associação Genética , Mutação , Fenótipo , Síndrome de Rubinstein-Taybi/diagnóstico , Síndrome de Rubinstein-Taybi/genética , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Criança , Pré-Escolar , Exoma , Éxons , Fácies , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto , Adulto Jovem
6.
J Am Soc Nephrol ; 25(9): 1942-53, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24676634

RESUMO

FSGS is characterized by the presence of partial sclerosis of some but not all glomeruli. Studies of familial FSGS have been instrumental in identifying podocytes as critical elements in maintaining glomerular function, but underlying mutations have not been identified for all forms of this genetically heterogeneous condition. Here, exome sequencing in members of an index family with dominant FSGS revealed a nonconservative, disease-segregating variant in the PAX2 transcription factor gene. Sequencing in probands of a familial FSGS cohort revealed seven rare and private heterozygous single nucleotide substitutions (4% of individuals). Further sequencing revealed seven private missense variants (8%) in a cohort of individuals with congenital abnormalities of the kidney and urinary tract. As predicted by in silico structural modeling analyses, in vitro functional studies documented that several of the FSGS-associated PAX2 mutations perturb protein function by affecting proper binding to DNA and transactivation activity or by altering the interaction of PAX2 with repressor proteins, resulting in enhanced repressor activity. Thus, mutations in PAX2 may contribute to adult-onset FSGS in the absence of overt extrarenal manifestations. These results expand the phenotypic spectrum associated with PAX2 mutations, which have been shown to lead to congenital abnormalities of the kidney and urinary tract as part of papillorenal syndrome. Moreover, these results indicate PAX2 mutations can cause disease through haploinsufficiency and dominant negative effects, which could have implications for tailoring individualized drug therapy in the future.


Assuntos
Glomerulosclerose Segmentar e Focal/genética , Mutação , Fator de Transcrição PAX2/genética , Adolescente , Adulto , Idade de Início , Idoso , Sequência de Aminoácidos , Sequência de Bases , Estudos de Coortes , Simulação por Computador , Sequência Conservada , Análise Mutacional de DNA , Exoma , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fator de Transcrição PAX2/química , Fator de Transcrição PAX2/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Eletricidade Estática , Anormalidades Urogenitais , Refluxo Vesicoureteral/genética , Adulto Jovem
7.
Front Mol Neurosci ; 16: 1170061, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37324589

RESUMO

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders.

8.
J Virol ; 85(10): 5070-80, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21389130

RESUMO

Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-ß signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-ß expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-ß and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.


Assuntos
Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/imunologia , Fator Regulador 1 de Interferon/metabolismo , Interferon beta/biossíntese , Queratinócitos/imunologia , Proteínas Oncogênicas Virais/metabolismo , Linhagem Celular , Papillomavirus Humano 16/patogenicidade , Humanos , Queratinócitos/virologia
9.
Nat Commun ; 13(1): 6841, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369169

RESUMO

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.


Assuntos
Transtornos do Neurodesenvolvimento , Peixe-Zebra , Humanos , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Complexo de Golgi/metabolismo , Retículo Endoplasmático/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo
10.
Diagn Microbiol Infect Dis ; 62(3): 280-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18801637

RESUMO

Ascaris presence in humans has been associated with high levels of blood eosinophils and serum IgE. This study was designed to address the influence of Ascaris infection on allergic and inflammatory parameters of atopic subjects. A cross-sectional design was used, and atopic individuals to be assessed were divided into 3 groups including Ascaris-infected, anti-Ascaris IgG-positive (seropositive), and control subjects. All subjects enrolled had positive skin test reactivity to at least 1 perennial or seasonal allergen; however, levels of C-reactive protein, C3, and C4 were within normal range values. Eosinophil percentage was not significantly different among the groups studied. Total IgE and specific anti-Ascaris IgE levels in the seropositive group were significantly higher than concentrations found in both control and infected groups. Interleukin (IL)-4 release in Ascaris-infected patients was significantly increased versus seropositives, who were able to produce more IL-4 than controls. The levels of IL-10 were lower in the seropositives as well as infected subjects in comparison with controls. CD25(+) lymphocyte populations were significantly increased in the infected group versus the seropositives as well as the controls. Lung function tests of some selected seropositive subjects were significantly impaired. The same parameters of a representative infected patient were not different from controls. Our data on T helper type 2 cells (Th2) and regulatory T cells (Treg) features, as well as CD25(+) lymphocyte increase, suggest an Ascaris-induced mechanism leading to parasite survival. Moreover, the stable control of both T helper type 1 cells (Th1) and Th2 immunity cascades, paralleled by the absence of overwhelming inflammatory systemic reactions and lack of allergic syndromes, may result in a favorable host condition.


Assuntos
Ascaríase/imunologia , Ascaris lumbricoides/imunologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/sangue , Subunidade alfa de Receptor de Interleucina-2/análise , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Idoso , Análise de Variância , Animais , Anticorpos Antiprotozoários/sangue , Estudos Transversais , Eosinófilos/imunologia , Fezes/parasitologia , Feminino , Humanos , Imunoglobulina E/metabolismo , Interferon gama/sangue , Interleucinas/sangue , Pessoa de Meia-Idade , Testes de Função Respiratória , Testes Cutâneos , Fator de Crescimento Transformador beta/sangue , Fator de Necrose Tumoral alfa/sangue
11.
Adv Med Sci ; 63(1): 87-93, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28957739

RESUMO

PURPOSE: Noonan Syndrome (NS) is an autosomal dominant disorder with many variable and heterogeneous conditions. The genetic basis for 20-30% of cases is still unknown. This study evaluates Iranian Noonan patients both clinically and genetically for the first time. MATERIALS/METHODS: Mutational analysis of PTPN11 gene was performed in 15 Iranian patients, using PCR and Sanger sequencing at phase one. Then, as phase two, Next Generation Sequencing (NGS) in the form of targeted resequencing was utilized for analysis of exons from other related genes. Homology modelling for the novel founded mutations was performed as well. The genotype, phenotype correlation was done according to the molecular findings and clinical features. RESULTS: Previously reported mutation (p.N308D) in some patients and a novel mutation (p.D155N) in one of the patients were identified in phase one. After applying NGS methods, known and new variants were found in four patients in other genes, including: CBL (p. V904I), KRAS (p. L53W), SOS1 (p. I1302V), and SOS1 (p. R552G). Structural studies of two deduced novel mutations in related genes revealed deficiencies in the mutated proteins. Following genotype, phenotype correlation, a new pattern of the presence of intellectual disability in two patients was registered. CONCLUSIONS: NS shows strong variable expressivity along the high genetic heterogeneity especially in distinct populations and ethnic groups. Also possibly unknown other causative genes may be exist. Obviously, more comprehensive and new technologies like NGS methods are the best choice for detection of molecular defects in patients for genotype, phenotype correlation and disease management.


Assuntos
Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Síndrome de Noonan/genética , Humanos , Irã (Geográfico) , Modelos Moleculares , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
12.
Neurology ; 91(4): e319-e330, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29959261

RESUMO

OBJECTIVE: To characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families. METHODS: We used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model. RESULTS: We identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years. CONCLUSIONS: This study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy.


Assuntos
Alelos , Progressão da Doença , Mutação/genética , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/genética , Proteína Sequestossoma-1/genética , Adolescente , Adulto , Idade de Início , Animais , Feminino , Humanos , Masculino , Linhagem , Sequenciamento do Exoma/métodos , Adulto Jovem , Peixe-Zebra
13.
Cancer Res ; 77(21): 5860-5872, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29092923

RESUMO

The basis for molecular and cellular heterogeneity in ependymomas of the central nervous system is not understood. This study suggests a basis for this phenomenon in the selection for mitogen-independent (MI) stem-like cells with impaired proliferation but increased intracranial tumorigenicity. MI ependymoma cell lines created by selection for EGF/FGF2-independent proliferation exhibited constitutive activation of EGFR, AKT, and STAT3 and sensitization to the antiproliferative effects of EGFR tyrosine kinase inhibitors (TKI). One highly tumorigenic MI line harbored membrane-bound, constitutively active, truncated EGFR. Two EGFR mutants (ΔN566 and ΔN599) were identified as products of intrachromosomal rearrangements fusing the 3' coding portion of the EGFR gene to the 5'-UTR of the SEC61G, yielding products lacking the entire extracellular ligand-binding domain of the receptor while retaining the transmembrane and tyrosine kinase domains. EGFR TKI efficiently targeted ΔN566/ΔN599-mutant-mediated signaling and prolonged the survival of mice bearing intracranial xenografts of MI cells harboring these mutations. RT-PCR sequencing of 16 childhood ependymoma samples identified SEC61G-EGFR chimeric mRNAs in one infratentorial ependymoma WHO III, arguing that this fusion occurs in a small proportion of these tumors. Our findings demonstrate how in vitro culture selections applied to genetically heterogeneous tumors can help identify focal mutations that are potentially pharmaceutically actionable in rare cancers. Cancer Res; 77(21); 5860-72. ©2017 AACR.


Assuntos
Ependimoma/genética , Receptores ErbB/genética , Canais de Translocação SEC/genética , Células-Tronco/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Criança , Células Clonais , Ependimoma/metabolismo , Ependimoma/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos Nus , Mitógenos/farmacologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Translocação SEC/metabolismo , Células-Tronco/efeitos dos fármacos , Transplante Heterólogo
14.
Nat Genet ; 46(8): 815-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25017102

RESUMO

Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism.


Assuntos
Anormalidades Múltiplas/genética , Calcinose/genética , Otopatias/genética , Deficiência Intelectual/genética , Atrofia Muscular/genética , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Deleção Cromossômica , Cromossomos Humanos Par 3 , Deficiências do Desenvolvimento/genética , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
15.
Clin Vaccine Immunol ; 16(12): 1804-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19828771

RESUMO

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvula is involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonella are very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvula lipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvula LPS stimulated tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-alpha and IL-6 production in PBMC stimulated with either Veillonella or Escherichia coli LPS. However, V. parvula LPS was 10- to 100-fold less active than E. coli LPS for cytokine induction. TNF-alpha, IL-1beta, IL-6, and IL-10 were released in wild-type and TLR2(-/-), but not TLR4(-/-), mouse macrophage cultures. V. parvula LPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvula LPS-induced TNF-alpha, IL-1beta, IL-6, and IL-10. In conclusion, V. parvula LPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than Enterobacteriaceae LPS. V. parvula LPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.


Assuntos
Leucócitos Mononucleares/imunologia , Macrófagos Peritoneais/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Veillonella/imunologia , Animais , Bartonella quintana/imunologia , Escherichia coli/imunologia , Humanos , Interleucina-10/agonistas , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-1beta/agonistas , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-6/agonistas , Interleucina-6/imunologia , Interleucina-6/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/agonistas , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Virol ; 81(1): 202-14, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17050603

RESUMO

Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects of nonstructural proteins are well established, the role of structural proteins remains controversial. We investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1), a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immunomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypeptide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may play a pivotal role in establishing persistent infection by dampening an effective immune response.


Assuntos
Hepacivirus/patogenicidade , Fator Regulador 1 de Interferon/antagonistas & inibidores , Proteínas do Core Viral/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Células Jurkat , Luciferases/análise , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Replicon , Transdução de Sinais , Transcrição Gênica
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