RESUMO
BACKGROUND: Research in recent years firmly established that microglial cells play an important role in the pathogenesis of Alzheimer's disease (AD). In parallel, a series of studies showed that, under both homeostatic and pathological conditions, microglia are a heterogeneous cell population. In AD, amyloid-ß (Aß) plaque-associated microglia (PAM) display a clearly distinct phenotype compared to plaque-distant microglia (PCM), suggesting that these two microglia subtypes likely differently contribute to disease progression. So far, molecular characterization of PAM was performed indirectly using single cell RNA sequencing (scRNA-seq) approaches or based on markers that are supposedly up-regulated in this microglia subpopulation. METHODS: In this study based on a well-characterized AD mouse model, we combined cell-specific laser capture microdissection and RNA-seq analysis to i) identify, without preconceived notions of the molecular and/or functional changes that would affect these cells, the genes and gene networks that are dysregulated in PAM or PCM at three critical stages of the disease, and ii) to investigate the potential contribution of both plaque-associated and plaque-distant microglia. RESULTS: First, we established that our approach allows selective isolation of microglia, while preserving spatial information and preventing transcriptome changes induced by classical purification approaches. Then, we identified, in PAM and PCM subpopulations, networks of co-deregulated genes and analyzed their potential functional roles in AD. Finally, we investigated the dynamics of microglia transcriptomic remodeling at early, intermediate and late stages of the disease and validated select findings in postmortem human AD brain. CONCLUSIONS: Our comprehensive study provides useful transcriptomic information regarding the respective contribution of PAM and PCM across the Aß pathology progression. It highlights specific pathways that would require further study to decipher their roles across disease progression. It demonstrates that the proximity of microglia to Aß-plaques dramatically alters the microglial transcriptome and reveals that these changes can have both positive and negative impacts on the surrounding cells. These opposing effects may be driven by local microglia heterogeneity also demonstrated by this study. Our approach leads to molecularly define the less well studied plaque-distant microglia. We show that plaque-distant microglia are not bystanders of the disease, although the transcriptomic changes are far less striking compared to what is observed in plaque-associated microglia. In particular, our results suggest they may be involved in Aß oligomer detection and in Aß-plaque initiation, with increased contribution as the disease progresses.
Assuntos
Doença de Alzheimer , Microglia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Placa Amiloide/patologia , TranscriptomaRESUMO
Brain banks manage and store fully clinically and pathologically characterised brains. The diversity of techniques used in research projects increases. These biological resource centres are made to adapt brain tissue processing. Furthermore, the development of more sensitive techniques to analyse nucleic acids and proteins offers new fields of exploration when combined with laser capture microdissection in order to decipher the physiopathology of diseases at the cell level. In this study, our goal was to evaluate procedures and set a workflow compatible with the constraints of brain banks, from brain sampling to laser capture microdissection and pre-analytical quality assessment. We compared various methods of freezing brain tissue, focused on morphological quality preservation of brain microscopical structures and on the quality of nucleic acid or protein yields. Staining protocols combined with strategies to lower neurones autofluorescence were adapted for the same purpose. Finally, we found that laser capture microdissection is possible in the setting of brain banks. However, the entire process has to be envisioned from the autopsy to the analysis. The impact on protein or nucleic acid quality is a limitation that restricts the amount of samples available for this purpose.
Assuntos
Encéfalo/patologia , Microdissecção , Neurônios/patologia , Bancos de Tecidos , Fluxo de Trabalho , Adulto , Idoso , Idoso de 80 Anos ou mais , Encefalopatias/patologia , Europa (Continente) , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Mudanças Depois da Morte , Proteínas/genética , Proteínas/metabolismo , Manejo de Espécimes , Coloração e RotulagemRESUMO
BACKGROUND: Hantaviruses are single-stranded RNA viruses, which are transmitted to humans primarily via inhalation of aerosolised virus in contaminated rodent urine and faeces. Whilst infected reservoir hosts are asymptomatic, human infections can lead to two clinical manifestations, haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with varying degrees of clinical severity. The incidence of rodent and human cases of Seoul virus (SEOV) in Europe has been considered to be low, and speculated to be driven by the sporadic introduction of infected brown rats (Rattus norvegicus) via ports. METHODS: Between October 2010 and March 2012, 128 brown rats were caught at sites across the Lyon region in France. RESULTS: SEOV RNA was detected in the lungs of 14% (95% CI 8.01-20.11) of brown rats tested using a nested pan-hantavirus RT-PCR (polymerase gene). Phylogenetic analysis supports the inclusion of the Lyon SEOV within Lineage 7 with SEOV strains originating from SE Asia and the previously reported French & Belgian SEOV strains. Sequence data obtained from the recent human SEOV case (Replonges) was most similar to that obtained from one brown rat trapped in a public park in Lyon city centre. We obtained significantly improved recovery of virus genome sequence directly from SEOV infected lung material using a simple viral enrichment approach and NGS technology. CONCLUSIONS: The detection of SEOV in two wild caught brown rats in the UK and the multiple detection of SEOV infected brown rats in the Lyon region of France, suggests that SEOV is circulating in European brown rats. Under-reporting and difficulties in identifying the hantaviruses associated with HFRS may mask the public health impact of SEOV in Europe.
Assuntos
Portador Sadio/veterinária , Reservatórios de Doenças , Ratos/virologia , Vírus Seoul/isolamento & purificação , Animais , Portador Sadio/epidemiologia , Portador Sadio/virologia , Análise por Conglomerados , França/epidemiologia , Pulmão/virologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/genética , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn 4 (-/-) ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn 4 (-/-) mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.
Assuntos
Relógios Circadianos , Dopamina/metabolismo , Regulação da Expressão Gênica , Luz , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Opsinas de Bastonetes/fisiologia , Animais , Ritmo Circadiano , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Circadianas Period/genética , Opsinas de Bastonetes/genética , Transdução de Sinais , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The choroid plexus epithelium controls the movement of solutes between the blood and the cerebrospinal fluid. It has been considered as a functionally more immature interface during brain development than in adult. The anatomical basis of this barrier is the interepithelial choroidal junction whose tightness has been attributed to the presence of claudins. We used quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry to identify different claudins in the choroid plexuses of developing and adult rats. Claudin-1, -2, and -3 were highly and selectively expressed in the choroid plexus as compared to brain or parenchyma microvessels and were localized at epithelial junctions. Claudin-6, -9, -19, and -22 also displayed a previously undescribed choroidal selectivity, while claudin-4, -5, and -16 were enriched in the cerebral microvessels. The choroidal pattern of tight junction protein expression in prenatal brains was already complex and included occludin and zonula occludens proteins. It differed from the adult pattern in that the pore-forming claudin-2, claudin-9, and claudin-22 increased during development, while claudin-3 and claudin-6 decreased. Claudin-2 and claudin-11 presented a mirror image of abundance between lateral ventricle and fourth ventricle choroid plexuses. Imunohistochemical analysis of human fetal and postnatal brains for claudin-1, -2, and -3 demonstrated their early presence and localization at the apico-lateral border of the choroid plexus epithelial cells. Overall, choroidal epithelial tight junctions are already complex in developing brain. The observed differences in claudin expression between developing and adult choroid plexuses may indicate developmental differences in selective blood-cerebrospinal fluid transport functions.
Assuntos
Barreira Hematoencefálica/metabolismo , Claudinas/análise , Claudinas/genética , Perfilação da Expressão Gênica , Animais , Western Blotting , Plexo Corióideo/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/metabolismoRESUMO
Context and Aim: Lipid overnutrition in female rabbits, from prepuberty, leads to impaired metabolism (dyslipidemia and increased adiposity) and follicular atresia, and, when continued during gestation, affects offspring phenotype with intrauterine growth retardation (IUGR) and leads to placental and lipid metabolism abnormalities. Growth retardation is already observed in embryo stage, indicating a possible implication of periconceptional exposure. The objective of this study was to discriminate the effects of preconception and gestational exposures on feto-placental development. Materials and Methods: Rabbit 1-day zygotes were collected from female donors under control (CD) or high-fat-high-cholesterol (HD) diet and surgically transferred to the left and right uterus, respectively, of each H (n = 6) or C (n = 7) synchronized recipients. Close to term, four combinations, CC (n = 10), CH (n = 13), HC (n = 13), and HH (n = 6), of feto-placental units were collected, for biometry analyses. Fatty acid (FA) profiles were determined in placental labyrinth, decidua, fetal plasma, and fetal liver by gas chromatography and explored further by principal component analysis (PCA). Candidate gene expression was also analyzed by RT-qPCR in the placenta and fetal liver. Data were analyzed by Kruskal-Wallis followed by Dunn's pairwise comparison test. Combinations of different data sets were combined and explored by multifactorial analysis (MFA). Results: Compared to controls, HH fetuses were hypotrophic with reduced placental efficiency and altered organogenesis, CH presented heavier placenta but less efficient, whereas HC presented a normal biometry. However, the MFA resulted in a good separation of the four groups, discriminating the effects of each period of exposure. HD during gestation led to reduced gene expression (nutrient transport and metabolism) and big changes in FA profiles in both tissues with increased membrane linoleic acid, lipid storage, and polyunsaturated-to-saturated FA ratios. Pre-conception exposure had a major effect on fetal biometry and organogenesis in HH, with specific changes in FA profiles (increased MUFAs and decreased LCPUFAs). Conclusion: Embryo origin left traces in end-gestation feto-placental unit; however, maternal diet during gestation played a major role, either negative (HD) or positive (control). Thus, an H embryo developed favorably when transferred to a C recipient (HC) with normal biometry at term, despite disturbed and altered FA profiles.
RESUMO
BRCA1 inactivation is a hallmark of familial breast cancer, often associated with aggressive triple negative breast cancers. BRCA1 is a tumor suppressor with known functions in DNA repair, transcription regulation, cell cycle control, and apoptosis. In the present study, we demonstrate that BRCA1 is also a translational regulator. We previously showed that BRCA1 was implicated in translation regulation. Here, we asked whether translational control could be a novel function of BRCA1 that contributes to its tumor suppressive activity. A combination of RNA-binding protein immunoprecipitation, microarray analysis, and polysome profiling, was used to identify the mRNAs that were specifically deregulated under BRCA1 deficiency. Western blot analysis allowed us to confirm at the protein level the deregulated translation of a subset of mRNAs. A unique and dedicated cohort of patients with documented germ-line BRCA1 pathogenic variant statues was set up, and tissue microarrays with the biopsies of these patients were constructed and analyzed by immunohistochemistry for their content in each candidate protein. Here, we show that BRCA1 translationally regulates a subset of mRNAs with which it associates. These mRNAs code for proteins involved in major programs in cancer. Accordingly, the level of these key proteins is correlated with BRCA1 status in breast cancer cell lines and in patient breast tumors. ADAT2, one of these key proteins, is proposed as a predictive biomarker of efficacy of treatments recently recommended to patients with BRCA1 deficiency. This study proposes that translational control may represent a novel molecular mechanism with potential clinical impact through which BRCA1 is a tumor suppressor.
Assuntos
Proteína BRCA1/genética , Neoplasias de Mama Triplo Negativas/genética , Adulto , Idoso , Proteína BRCA1/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Pessoa de Meia-Idade , Transfecção , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Saethre-Chotzen syndrome (SCS), also known as acrocephalosyndactyly III, is an autosomal dominant hereditary disorder characterized by craniofacial and limb anomalies. SCS is generally caused by mutations in the TWIST gene, but several 7p21.3 microdeletions involving the entire gene have also been described. The patient reported here presented with craniosynostosis, ptosis, brachydactyly and syndactyly of toes. Standard lymphocyte karyotype showed a de novo apparently balanced but complex constitution with a translocation between the short arms of chromosomes 2 and 7 and an insertion of the 7(q21.3q22) band in the short arm of the same chromosome 7. Interestingly, array CGH displayed a unique 690 kb deletion in 7p21.3 involving the TWIST gene, consistent with the phenotype. This case illustrates the important contribution of array CGH to identification of complex chromosomal rearrangements.
Assuntos
Acrocefalossindactilia/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 7/genética , Deleção de Genes , Rearranjo Gênico , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Acrocefalossindactilia/patologia , Adulto , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
Although most pituitary tumors are benign, some are invasive or aggressive. In the absence of specific markers of malignancy, only tumors with metastases are considered malignant. To identify markers of invasion and aggressiveness, we focused on prolactin (PRL) tumors in the human and rat. Using radiology and histological methods, we classified 25 human PRL tumors into three groups (non-invasive, invasive, and aggressive-invasive) and compared them with a model of transplantable rat PRL tumors with benign and malignant lineages. Combining histological(mitoses and labeling for Ki-67, P53, pituitary transforming tumor gene (PTTG), and polysialic acid neural cell adhesion molecule) and transcriptomic (microarrays and q-RTPCR) methods with clinical data (post-surgical outcome with case-control statistical analysis), we found nine genes implicated in invasion (ADAMTS6, CRMP1, and DCAMKL3) proliferation (PTTG, ASK, CCNB1, AURKB, and CENPE), or pituitary differentiation (PITX1) showing differential expression in the three groups of tumors (P = 0.015 to 0.0001). A case-control analysis, comparing patients in remission (9 controls) and patients with persistent or recurrent tumors (14 cases) revealed that eight out of the nine genes were differentially up- or downregulated (P = 0.05 to 0.002), with only PTTG showing no correlation with clinical course (P = 0.258). These combined histological and transcriptomic analyses improve the pathological diagnosis of PRL tumors, indicating a reliable procedure for predicting tumor aggressiveness and recurrence potential. The similar gene profiles found between non-invasive human and benign rat tumors, as well as between aggressive-invasive human and malignant rat tumors provide new insights into malignancy in human pituitary tumors.
Assuntos
Biomarcadores Tumorais/isolamento & purificação , Proliferação de Células , Técnicas de Diagnóstico Molecular , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Prolactinoma/genética , Prolactinoma/patologia , Animais , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Invasividade Neoplásica , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Hipofisárias/classificação , Neoplasias Hipofisárias/diagnóstico , Prognóstico , Prolactinoma/classificação , Prolactinoma/diagnóstico , Ratos , Ratos WistarRESUMO
Changes in microRNAs (miRNAs) expression in many types of cancer suggest that they may be involved in crucial steps during tumor progression. Indeed, miRNAs deregulation has been described in pituitary tumorigenesis, but few studies have described their role in pituitary tumor progression toward aggressiveness and malignancy. To assess the role of miRNAs within the hierarchical cascade of events in prolactin (PRL) tumors during progression, we used an integrative genomic approach to associate clinical-pathological features, global miRNA expression, and transcriptomic profiles of the same human tumors. We describe the specific down-regulation of one principal miRNA, miR-183, in the 8 aggressive (A, grade 2b) compared to the 18 non-aggressive (NA, grades 1a, 2a) PRL tumors. We demonstrate that it acts as an anti-proliferative gene by directly targeting KIAA0101, which is involved in cell cycle activation and inhibition of p53-p21-mediated cell cycle arrest. Moreover, we show that miR-183 and KIAA0101 expression significantly correlate with the main markers of pituitary tumors aggressiveness, Ki-67 and p53. These results confirm the activation of proliferation in aggressive and malignant PRL tumors compared to non-aggressive ones. Importantly, these data also demonstrate the ability of such an integrative genomic strategy, applied in the same human tumors, to identify the molecular mechanisms responsible for tumoral progression even from a small cohort of patients.
RESUMO
We recently reported that immature porcine Leydig cells express both somatostatin (SRIF) and SRIF receptor type-2 (sst-2) transcripts. The present study was therefore undertaken to assess whether SRIF might exert autocrine actions on these cells through sst2A receptor, one of the two sst2 isoforms known to exert important neuroendocrine and endocrine functions. Using a polyclonal antibody directed towards the C-terminal tail of the sst2A receptor subtype, receptor immunoreactivity was detected in a subpopulation of Leydig cells and spermatogonia. To address the physiological correlates of this expression we then studied the possible involvement of sst2 receptor in the regulation of testosterone secretion. Functional assays showed that the sst2 agonist octreotide inhibited both basal and hCG-stimulated testosterone secretion by testosterone pretreated Leydig cells. To assess whether sst2 receptor expression might be regulated by testosterone, we performed a semi-quantitative RT-PCR analysis of sst2 mRNA expression in Leydig cells cultured in the presence or in the absence of the androgen. A significant increase in sst2 receptor transcripts was observed in testosterone-treated cells. Taken together, these data suggest that SRIF can inhibit testosterone secretion through the sst2A receptor. The mechanism of the local inhibitory actions of SRIF is probably autocrine since immature porcine Leydig cells express SRIF itself and it might involve testosterone-induced increase of sst2 receptor expression in immature Leydig cells.
Assuntos
Células Intersticiais do Testículo/metabolismo , Receptores de Somatostatina/biossíntese , Somatostatina/fisiologia , Suínos/fisiologia , Testosterona/metabolismo , Animais , Comunicação Autócrina , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Gonadotropina Coriônica/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Octreotida/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Somatostatina/genética , Receptores de Somatostatina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/agonistas , Somatostatina/biossíntese , Testículo/citologia , Testículo/crescimento & desenvolvimentoRESUMO
THE INFLAMMATORY RESPONSE FOLLOWING ISCHEMIC STROKE IS DOMINATED BY INNATE IMMUNE CELLS: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.
Assuntos
Isquemia Encefálica/imunologia , Isquemia Encefálica/terapia , Macrófagos/imunologia , Pesquisa Translacional Biomédica , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Morte Celular/imunologia , Hipóxia Celular/imunologia , Modelos Animais de Doenças , Hipocampo/imunologia , Hipocampo/patologia , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/imunologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/terapia , Macrófagos/citologia , Masculino , Camundongos , Neurônios/patologia , Ratos , Acidente Vascular Cerebral/complicações , Resultado do TratamentoRESUMO
Integrative genomics approaches associating DNA structure and transcriptomic analysis should allow the identification of cascades of events relating to tumor aggressiveness. While different genome alterations have been identified in pituitary tumors, none have ever been correlated with the aggressiveness. This study focused on one subtype of pituitary tumor, the prolactin (PRL) pituitary tumors, to identify molecular events associated with the aggressive and malignant phenotypes. We combined a comparative genomic hybridization and transcriptomic analysis of 13 PRL tumors classified as nonaggressive or aggressive. Allelic loss within the p arm region of chromosome 11 was detected in five of the aggressive tumors. Allelic loss in the 11q arm was observed in three of these five tumors, all three of which were considered as malignant based on the occurrence of metastases. Comparison of genomic and transcriptomic data showed that allelic loss impacted upon the expression of genes located in the imbalanced region. Data filtering allowed us to highlight five deregulated genes (DGKZ, CD44, TSG101, GTF2H1, HTATIP2), within the missing 11p region, potentially responsible for triggering the aggressive and malignant phenotypes of PRL tumors. Our combined genomic and transcriptomic analysis underlines the importance of chromosome allelic loss in determining the aggressiveness and malignancy of tumors.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 11 , Neoplasias Hipofisárias/genética , Prolactinoma/genética , Acetiltransferases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Ciclo Celular/genética , Hibridização Genômica Comparativa , Proteínas de Ligação a DNA/genética , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Regulação para Baixo/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Feminino , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença/genética , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , Transdução de Sinais/genética , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição TFII/genéticaRESUMO
Human pineal parenchymal tumors (PPTs) are rare tumors, and little is known about their molecular pathogenesis. We used Atlas plastic human 8 K microarray analysis to identify the genes expressed in four human PPTs of different grades, in normal brain tissue and in a normal fetal pineal gland. We selected the most highly expressed genes in PPT (n=39) and compared their expression to that both in normal brain and fetal pineal gland. Nine genes were expressed more than twice as strongly and 3 at about half the level in PPT. Furthermore, real-time reverse transcription-PCR was performed to compare mRNA levels in the four PPTs, in four medulloblastomas (MBs) (the most common type of similar embryonal neoplasm in the cerebellum), and in normal brain, for 9 of the 39 genes. Among genes showing an expression similar to that obtained with microarray, puromycin-sensitive aminopeptidase and teratocarcinoma-derived growth factor 3 were up-regulated in PPT and in MB, and adenomatous polyposis coli like was down-regulated only in PPT. Up-regulated expression of chromosome 17 open reading frame 1A was seen in high-grade PPT and in MB, but not in lower grade PPT. In conclusion, our results identified a number of genes that are differentially expressed in PPT and MB, and some of them may serve as prognostic markers and can be used to define mechanisms of tumorigenesis.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Expressão Gênica/fisiologia , Análise em Microsséries/métodos , Glândula Pineal , Pinealoma/genética , Neoplasias Encefálicas/metabolismo , Feminino , Feto , Humanos , Masculino , Pinealoma/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor alpha suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor alpha-induced apoptosis.