RESUMO
Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies.
Assuntos
Transtornos Cognitivos/fisiopatologia , Hipocampo/fisiopatologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptor A2A de Adenosina/metabolismo , Tauopatias/fisiopatologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Animais , Transtornos Cognitivos/tratamento farmacológico , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Humanos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos Transgênicos , Fosforilação , RNA Mensageiro/metabolismo , Receptor A2A de Adenosina/genética , Tauopatias/tratamento farmacológico , Técnicas de Cultura de Tecidos , Xantinas/farmacologia , Ácido gama-Aminobutírico/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele â¼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Predisposição Genética para Doença/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Proteínas tau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endofenótipos , Expressão Gênica/genética , Humanos , Camundongos , Degeneração Neural/genética , Degeneração Neural/patologia , Proteínas Nucleares/biossíntese , Placa Amiloide/patologia , Polimorfismo de Nucleotídeo Único/genética , Sinaptossomos/patologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas tau/antagonistas & inibidoresRESUMO
The peptidyl prolyl cis-trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr-Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.
Assuntos
Regulação para Baixo , Proteínas Inibidoras de Apoptose/metabolismo , Peptidilprolil Isomerase/metabolismo , Animais , Sítios de Ligação , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA Complementar , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Peptidilprolil Isomerase/genética , Testes de Precipitina , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The chimeric tyrosine kinase p210BCR-ABL is involved in the pathogenesis of chronic myelogenous leukemia. It transforms immature hematopoietic cells in vitro and abrogates IL-3-dependent growth. The mechanisms by which p210BCR-ABL mediates its oncogenicity are not well elucidated. Identifying transcription factors targeted by the chimeric protein may help to clarify these mechanisms. We have analysed the effect of p210BCR-ABL expression on NF-kappaB activity in DA1 cells (an IL-3-dependent murine myeloid progenitor cell line). A specific stimulation of NF-kappaB activity by kinase-active wild-type p210BCR-ABL has been evidenced by transcriptional activation assays. Electrophoretic mobility supershift assays revealed the presence of p65 protein (RelA) DNA binding activity in p210BCR-ABL transformed DA1 cells but not in parental DA1 cells. Activation of RelA in transformed DA1 cells may occur by protein stabilization. Experiments using oligonucleotides antisense to RelA showed that p210BCR-ABL transfected cells failed to survive after IL-3 removal. Moreover, inhibition of cellular growth was shown following treatment of p210BCR-ABL transformed DA1 cells by p65 antisense oligonucleotides. This study suggests that p65 NF-kappaB may be an effector for p210BCR-ABL and probably contributes to its induced transformation process.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Fusão bcr-abl/farmacologia , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Animais , Células da Medula Óssea , Linhagem Celular , Linhagem Celular Transformada , Interleucina-3/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , NF-kappa B/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oligonucleotídeos Antissenso , Oncogenes , RNA Mensageiro/análise , Sinaptotagmina I , Sinaptotagminas , Fator de Transcrição RelARESUMO
The hepatitis B virus (HBV) enhancer contains multiple active elements, one of which is the EP element, a 15 bp site important for its regulation by acting on other functional elements like the E site. The EP element, in the HBV enhancer context, contains two putative binding sites for c-myb family gene products. Electrophoretic mobility shift assays showed that the minimal c-Myb DNA-binding domain binds to the EP sequence. DNase I footprinting experiments revealed that only one consensus binding site was effectively protected. We found that c-Myb down-regulates transcription driving by the HBV enhancer in CAT assays performed in a haematopoietic (K562) and in a hepatic (HepG2) cell line. Interestingly, co-expression of both c-Myb and NF-M, a C/EBPbeta homologue which recognises the E element of the HBV enhancer, showed a synergistic transactivation of the HBV enhancer while, separately, each of them had an inhibitory effect on transcription in HepG2 and K562 cell lines, two cell types potentially infected by the hepatitis B virus.
Assuntos
Elementos Facilitadores Genéticos , Vírus da Hepatite B/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Pegada de DNA , Regulação para Baixo , Humanos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myb , Transativadores/química , Transativadores/genética , Transcrição GênicaRESUMO
Increasing evidence suggests that an inhibition of the proteasome, as demonstrated in Parkinson's disease, might be involved in Alzheimer's disease. In this disease and other Tauopathies, Tau proteins are hyperphosphorylated and aggregated within degenerating neurons. In this state, Tau is also ubiquitinated, suggesting that the proteasome might be involved in Tau proteolysis. Thus, to investigate if proteasome inhibition leads to accumulation, hyperphosphorylation and aggregation of Tau, we used neuroblastoma cells overexpressing Tau proteins. Surprisingly, we showed that the inhibition of the proteasome led to a bidirectional degradation of Tau. Following this result, the cellular mechanisms that may degrade Tau were investigated.
Assuntos
Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas tau/metabolismo , Anticorpos Fosfo-Específicos/imunologia , Caspases/análise , Caspases/metabolismo , Extratos Celulares/química , Linhagem Celular Tumoral , Humanos , Leupeptinas/farmacologia , Fosforilação , Poli(ADP-Ribose) Polimerases/análise , Poli(ADP-Ribose) Polimerases/metabolismo , Complexo de Endopeptidases do Proteassoma/análise , Inibidores de Proteassoma , Proteínas tau/análiseRESUMO
Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.
Assuntos
Estro/fisiologia , Hormônio Liberador de Gonadotropina/fisiologia , Eminência Mediana/fisiologia , Óxido Nítrico Sintase/fisiologia , Animais , Vasos Sanguíneos/fisiologia , Endotélio Vascular/fisiologia , Feminino , Eminência Mediana/irrigação sanguínea , Óxido Nítrico Sintase Tipo III , Ratos , Ratos WistarRESUMO
c-Abl tyrosine kinase, an essential protein of the cell cycle signalling pathways, is implicated in the regulation of RNA polymerase II activity, apoptosis and DNA repair. Its DNA binding activity is important for its biological functions. However, the molecular basis of c-Abl interaction with DNA remains largely unclear. We delimited the human c-Abl DNA binding domain and identified its preferred binding site, 5'-A(A/C)AACAA(A/C). The central AAC motif is highly conserved and constitutes the major core element in the binding sites. EMSAs and footprinting experiments were performed to explore how the c-Abl fusion protein recognizes specific sequences in DNA.
Assuntos
DNA/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Pegada de DNA , Desoxirribonuclease I/metabolismo , Eletroforese/métodos , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Software , Especificidade por SubstratoRESUMO
In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments. These Tau variants displayed specific epitopes that are immunoreactive with anti-phospho-Tau antibodies such as AT100. As shown in in vitro experiments, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase A (PKA) may be key kinases in these phosphorylation events. In the present study, Tau was microinjected into Xenopus oocytes. Surprisingly, in this system, AT100 was generated without any GSK3beta and PKA contribution during the progesterone or insulin-induced maturation process. Our results demonstrate that a non-modified physiological process in a cell model can generate the most specific Alzheimer epitope of Tau pathology.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Técnicas In Vitro , Cloreto de Lítio/farmacologia , Modelos Biológicos , Oócitos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , XenopusRESUMO
PURPOSE: To examine the CD40 costimulatory molecule expression on normal resting or activated adult human retinal pigment epithelium (hRPE) cells and to evaluate its role as an activation molecule considering the potential antigen presentation functions of hRPE cells. METHODS: Expression of HLA-DR and costimulatory (CD40, B7.1, B7.2, CD54, and CD58) molecules on hRPE cells was analyzed by flow cytometry. CD40 triggering was performed using soluble CD40L or cocultures with CD40L transfected fibroblasts. Interleukin (IL)-6, -8, -10, and -12 secretions were measured by enzyme-linked immunosorbent assay. Antigen presentation function of hRPE cells was assessed by coculturing hRPE cells with allogeneic T cells. T-cell proliferation was measured by [(3)H]-thymidine incorporation, and T-cell apoptosis by measurement of caspase-3 activity. RESULTS: Interferon (IFN)gamma-activated hRPE cells expressed CD40, but not B7.1 or B7.2. Although interferongamma enhanced IL-6 and IL-8 production, CD40 triggering of IFNgamma-activated hRPE cells did not induce IL-12 secretion. hRPE cells did not stimulate allogeneic resting T cells and downregulated phytohemagglutinin-activated allogeneic T cells via a cell-to-cell contact-dependent mechanism. Some induction of apoptosis was detected. CONCLUSIONS: CD40 is expressed on IFNgamma-activated hRPE cells. Its ligation leads to an increased production of IL-6 and IL-8 but fails to induce B7.1 or B7. 2 expression, or to induce IL-12 secretion. Accordingly, hRPE cells do not activate allogenic T cells but inhibit T-cell proliferation, partly through induction of apoptosis. These results suggest that hRPE cells could be implicated more in a deviant antigen presentation. If the exact molecular mechanisms are unclear, it is likely that CD40-CD40L interaction could play a role in this process.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Antígenos CD40/biossíntese , Epitélio Pigmentado Ocular/metabolismo , Animais , Apresentação de Antígeno/fisiologia , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Antígenos CD/biossíntese , Apoptose , Ligante de CD40 , Caspase 3 , Caspases/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Fibroblastos , Citometria de Fluxo , Antígenos HLA-DR/biossíntese , Humanos , Interferon gama/farmacologia , Ativação Linfocitária/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Linfócitos T/fisiologia , Regulação para CimaRESUMO
In tauopathies, comparative biochemistry of tau aggregates shows that they differ in both phosphorylation and content of tau isoforms. Six tau isoforms are found in human brain that contain either three (3R) or four microtubule-binding domains (4R). In Alzheimer's disease, all six of the tau isoforms are phosphorylated and aggregate into paired helical filaments. They are detected by immunoblotting as a major tau triplet (tau 55, 64, and 69). In corticobasal degeneration and progressive supranuclear palsy, only phosphorylated 4R-tau isoforms aggregate and appear as a major tau doublet (tau 64 and 69). In Pick's disease, only phosphorylated 3R-tau isoforms aggregate into filaments and are characterized by another major tau doublet (tau 55 and 64). Finally, recent findings provide a direct link between a genetic defect in tau and its abnormal aggregation into filaments in frontotemporal dementia with parkinsonism linked to chromosome 17. In the present study, the question of a relationship between tau isoforms and cell morphology is raised. To answer this question, stably transfected human neuroblastoma SY5Y cell lines with either 3R- or 4R-tau isoforms are established. Cell morphology and tau phosphorylation were modified, suggesting that cells undergo profound changes in their metabolism and viability.
Assuntos
Encéfalo/patologia , Mutação , Doenças Neurodegenerativas/genética , Neurônios/patologia , Polimorfismo Genético , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Cromossomos Humanos Par 17 , Humanos , Doenças Neurodegenerativas/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fenótipo , Fosforilação , Doença de Pick/genética , Doença de Pick/patologia , Isoformas de Proteínas/genética , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/patologia , Proteínas tau/químicaRESUMO
The Ewing-like variation of adamantinoma is a rare entity, leading to challenge its differential diagnosis, notably with Ewing's sarcoma. We are reporting a case of a 20-year-old male who presented with swelling in the left leg that had progressed over a 2-year period. X-rays revealed a tumour in the tibia that was intracortical, osteolytic, multilocular and invaded the soft tissues. A surgical biopsy was performed. Histopathology examination showed a tumour growth with small round cells expressing CD99. A diagnosis of Ewing's sarcoma was made. Since the patient declined surgical treatment, chemotherapy was administered. Two years later, the patient returned because the tumour had grown in size. A second biopsy was performed. Microscopic evaluation showed a tumour growth with osteofibrous and epithelial components, which expressed pankeratin and vimentin, but was negative for CD99. A diagnosis of Ewing-like adamantinoma was made.
Assuntos
Adamantinoma/diagnóstico , Sarcoma de Ewing/diagnóstico , Tíbia , Adamantinoma/terapia , Diagnóstico Diferencial , Humanos , Masculino , Adulto JovemRESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder histologically defined by the cerebral accumulation of amyloid deposits and neurofibrillary tangles composed of hyperphosphorylated tau proteins. Loss of basal forebrain cholinergic neurons is another hallmark of the disease thought to contribute to the cognitive dysfunctions. To this date, the mechanisms underlying cholinergic neurons degeneration remain uncertain. The present study aimed to investigate the relationship between neurofibrillary degeneration and cholinergic defects in AD using THY-Tau22 transgenic mouse model exhibiting a major hippocampal AD-like tau pathology and hyperphosphorylated tau species in the septohippocampal pathway. Here, we report that at a time THY-Tau22 mice display strong reference memory alterations, the retrograde transport of fluorogold through the septohippocampal pathway is altered. This impairment is associated with a significant reduction in the number of choline acetyltransferase (ChAT)-immunopositive cholinergic neurons in the medial septum. Analysis of nerve growth factor (NGF) levels supports an accumulation of the mature neurotrophin in the hippocampus of THY-Tau22 mice, consistent with a decrease of its uptake or retrograde transport by cholinergic terminals. Finally, our data strongly support that tau pathology could be instrumental in the cholinergic neuronal loss observed in AD.
Assuntos
Encéfalo/patologia , Neurônios Colinérgicos/patologia , Emaranhados Neurofibrilares/patologia , Proteínas tau/metabolismo , Animais , Encéfalo/metabolismo , Neurônios Colinérgicos/metabolismo , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Camundongos , Camundongos Transgênicos , Emaranhados Neurofibrilares/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas tau/genéticaRESUMO
The c-Abl tyrosine kinase protein is implicated in the signaling pathway as well as in transcription, DNA repair, apoptosis, and several other vital biological processes essential for cell proliferation or differentiation. The interaction of c-Abl with DNA is important for some of these functions, but the exact nature of this interaction is still a matter of controversy. The present study addresses the DNA-binding properties of the human c-Abl protein. Using CASTing experiments, the consensus binding site 5'-AA/CAACAAA/C was determined. The central highly conserved AAC triplet appears to constitute the crucial core element in the binding sequences of the c-Abl protein. The c-Abl DNA-binding domain recognizes specific sequences and interacts with deformed DNA structures such as four-way junctions and bubble DNA containing a large single-stranded loop, as determined by electromobility shift assay, melting temperature studies, and binding to specific oligonucleotides covalently linked to beads. Additional competition experiments suggest that the interaction mainly involves contacts within the minor groove of the double helix. The DNA-binding properties of c-Abl are reminiscent of those of high-mobility group (HMG)-like proteins such as LEF-1 and SRY. However, the circular permutation and ring closure assays and DNA unwinding experiments reveal that, unlike HMGs, c-Abl does not bend its target sequence. In addition, it is shown that the protein potentiates the DNA relaxation activity of topoisomerase I. These findings indicate that the interaction of c-Abl with DNA is both sequence-selective and structure-dependent.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Composição de Bases , Sequência de Bases , Sítios de Ligação/genética , DNA Topoisomerases Tipo I/farmacologia , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/efeitos dos fármacos , DNA Super-Helicoidal/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico , Proteínas Tirosina Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/farmacologia , TemperaturaRESUMO
Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.
Assuntos
Ativinas/genética , Ativinas/metabolismo , Subunidades beta de Inibinas/genética , Subunidades beta de Inibinas/metabolismo , Inibinas/genética , Inibinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Testículo/metabolismo , Adulto , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Células de Sertoli/metabolismo , Espermatogênese , Testículo/citologiaRESUMO
Transduction efficiency of different types of recombinant (r)AAV-2 based vectors preparations markedly differed, with apparently no correlation with the replicative titers. Using HeLa cells as target for transduction, 105 and 30 infectious units were necessary to observe one transductant using respectively cesium-chloride-purified rAAV and crude lysates of producer cells obtained by sonication. The purified vectors were however able to transduce HEK-193 cells efficiently, but transgene expression was detected with some delay compared with crude lysates. The unexpected high transduction efficiency of sonicated crude lysates was due to virally mediated gene transfer, since similar sonicated crude lysates, but with no AAV rep and cap genes, did not lead to detection of transgene products after incubation with HeLa cells. Furthermore, sonicated cellular extracts of 293 or 293/T cells given in trans stimulate transduction of HeLa cells by purified rAAV. In contrast, neither extracts from the adenovirus E1-transformed 911 cell line, nor from other cell lines not harboring any adenovirus gene, had enhancing effect on rAAV-mediated transduction. These data suggest that 293 sonicated extracts contain factors which stimulate rAAV-mediated transduction of cells that are normally poorly transduced and offer a system to identify such factors and to characterize further the steps limiting the transfer of gene by AAV vectors.