RESUMO
The biology of transposable elements (TEs) is a fascinating and complex field of investigation. TEs represent a substantial fraction of many eukaryotic genomes and can influence many aspects of DNA function that range from the evolution of genetic information to duplication, stability, and gene expression. Their ability to move inside the genome has been largely recognized as a double-edged sword, as both useful and deleterious effects can result. A fundamental role has been played by the evolution of the molecular processes needed to properly control the expression of TEs. Today, we are far removed from the original reductive vision of TEs as "junk DNA", and are more convinced that TEs represent an essential element in the regulation of gene expression. In this review, we summarize some of the more recent findings, mainly in the animal kingdom, concerning the active roles that TEs play at every level of gene expression regulation, including chromatin modification, splicing, and protein translation.
Assuntos
Elementos de DNA Transponíveis/genética , Animais , Regulação da Expressão Gênica/genética , HumanosRESUMO
EGR1 is a transcription factor expressed in many cell types that regulates genes involved in different biological processes including growth, proliferation, and apoptosis. Dysregulation of EGR1 expression has been associated with many pathological conditions such as tumors and brain diseases. Known molecular mechanisms underlying the control of EGR1 function include regulation of transcription, mRNA and protein stability, and post-translational modifications. Here we describe the identification of a splicing isoform for the human EGR1 gene. The newly identified splicing transcript encodes a shorter protein compared to the canonical EGR1. This isoform lacks a region belonging to the N-terminal activation domain and although it is capable of entering the nucleus, it is unable to activate transcription fully relative to the canonical isoform.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Processamento Alternativo , Linhagem Celular , Regulação da Expressão Gênica , Células HEK293 , Humanos , Isoformas de Proteínas/genética , RNA Mensageiro/genéticaRESUMO
Cortical spreading depression (CSD) is an evolutionarily conserved phenomenon that involves a slow and self-propagating depolarization wave associated with spontaneous depression of electrical neuronal activity. CSD plays a central role in the pathophysiology of several brain diseases and is considered to be able to promote "Preconditioning". This phenomenon consists of the brain protecting itself against future injury by adaptation. Understanding of the molecular mechanisms underlying Preconditioning has significant clinical implications. We have already proposed that the long-lasting effects of CSD could be related to silencing of retrotransposon sequences by histone methylation. We analyzed DNA methylation of two retrotransposon sequences, LINE1 and L1, and their corresponding expression pattern after CSD induction. Based on immunoprecipitation assay of the methylated DNA (meDIP), we demonstrated hypermethylation of both sequences in preconditioned rat brain cortex compared with a control 24 h after CSD induction. Using quantitative PCR, we also showed that CSD induction caused a decrease of the transcript level of both retrotransposon sequences. Our data are consistent with the hypothesis of epigenetic modifications in Preconditioning-dependent neuroprotection by increasing genome stability via the silencing of retrotransposon sequences.
Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Epigênese Genética , Elementos Nucleotídeos Longos e Dispersos , Animais , Metilação de DNA , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Óxido Nítrico Sintase Tipo II/genética , Fatores de Proteção , Ratos WistarRESUMO
The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Fatores de Transcrição Onecut/metabolismo , Células Fotorreceptoras/fisiologia , Elementos Reguladores de Transcrição/genética , Urocordados/genética , Animais , Eletroporação , Histocitoquímica , Hibridização In Situ , Itália , Mar Mediterrâneo , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/embriologia , Fatores de Transcrição Onecut/genética , Células Fotorreceptoras/metabolismo , TranscriptomaRESUMO
BACKGROUND: The Hox family of transcription factors has a fundamental role in segmentation pathways and axial patterning of embryonic development and their clustered organization is linked with the regulatory mechanisms governing their coordinated expression along embryonic axes. Among chordates, of particular interest are the Hox paralogous genes in groups 1-4 since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed. RESULTS: To investigate the degree of conservation in cis-regulatory components that form the basis of Hox expression in the anterior nervous system, we have used assays for transcriptional activity in ascidians and vertebrates to compare and contrast regulatory potential. We identified four regulatory sequences located near the CiHox1, CiHox2 and CiHox4 genes of the ascidian Ciona intestinalis which direct neural specific domains of expression. Using functional assays in Ciona and vertebrate embryos in combination with sequence analyses of enhancer fragments located in similar positions adjacent to Hox paralogy group genes, we compared the activity of these four Ciona cis-elements with a series of neural specific enhancers from the amphioxus Hox1-3 genes and from mouse Hox paralogous groups 1-4. CONCLUSIONS: This analysis revealed that Kreisler and Krox20 dependent enhancers critical in segmental regulation of the hindbrain appear to be specific for the vertebrate lineage. In contrast, neural enhancers that function as Hox response elements through the action of Hox/Pbx binding motifs have been conserved during chordate evolution. The functional assays reveal that these Hox response cis-elements are recognized by the regulatory components of different and extant species. Together, our results indicate that during chordate evolution, cis-elements dependent upon Hox/Pbx regulatory complexes, are responsible for key aspects of segmental Hox expression in neural tissue and appeared with urochordates after cephalochordate divergence.
Assuntos
Ciona intestinalis/embriologia , Ciona intestinalis/genética , Elementos Facilitadores Genéticos , Evolução Molecular , Genes Homeobox , Proteínas de Homeodomínio/genética , Animais , Embrião de Galinha , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
Sialic acid-binding immunoglobulin-type lectins, which are predominantly expressed in immune cells, represent a family of immunomodulatory receptors with inhibitory and activating signals, in both healthy and disease states. Genetic factors are important in all forms of dementia, especially in early onset dementia. CD33 was recently recognized as a genetic risk factor for Alzheimer disease (AD). Here, we present a 2-generation family with 4 members, the father and the 3 siblings, characterized by an early form of unusual dementia exhibiting a behavioral variant close to behavioral variant frontotemporal dementia phenotype and severe forms of memory loss suggestive of AD. We analyzed the CD33 gene in this family and identified 10 single nucleotide polymorphisms (SNPs) in a linkage disequilibrium block associated with the disease. We also identified a tag SNP, rs2455069-A>G, in CD33 exon 2 that could be involved with dementia risk. Additionally, we excluded the presence of C9orf72 expansion mutations and other mutations previously associated with sporadic FTD and AD. The tag SNP association was also analyzed in selected sporadic AD patients from the same Southern Italy region. We demonstrate that CD33 and SIGLECL1 have a significantly increased level of expression in these patients.
Assuntos
Demência/genética , Predisposição Genética para Doença/genética , Lectinas/genética , Proteínas de Membrana/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Self-association of histones H1 from calf thymus and from sperm of the marine worm Chaetopterus variopedatus was studied on native and glutaraldehyde cross-linked molecules by PAGE and by salt-induced turbidity measurements. Multiple polymers were generated by native sperm histone H1-like after glutaraldehyde cross-linking while the same treatment on its lysine- or arginine-modified derivatives and on somatic histone H1 failed to induce polymerization. This result suggests the relevance of arginine content in the formation of histone H1-like polymers particularly because Chaetopterus variopedatus and calf thymus histones H1 have similar content of lysine but different K/R ratio (2 and 15, respectively). Salt-induced turbidity experiments confirmed the high tendency of sperm histone H1-like to form oligomers, particularly in the presence of phosphate ions. Native PAGE analysis in the presence of phosphate supported this hypothesis. The reported results suggest that phosphate ions connecting lysine and arginine side chain groups contribute to the interaction of sperm histone H1-like with DNA in chromatin and play a key role in organization and stabilization of the chromatin higher order structures.
Assuntos
Arginina/metabolismo , Histonas/metabolismo , Poliquetos/metabolismo , Espermatozoides/metabolismo , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Histonas/química , Histonas/fisiologia , MasculinoRESUMO
Progranulin (GRN) gene mutations have been genetically associated with frontotemporal dementia (FTD) and are present in about 23% of patients with familial FTD. However, the neurobiology of this secreted glycoprotein remains unclear. Here, we report the identification of 3 pedigrees of Southern Italian extraction in whom FTD segregates with autosomal dominant inheritance patterns. We present evidence that all the available patients in these 3 familial cases are carrying the rare GRN gene exon 6 deletion g10325_10331delCTGCTGT (relative to nt 1 inNG_007886.1), alias Cys157LysfsX97. This mutation was previously described in 2 sporadic cases but was never associated with familial cases. Our patients demonstrate heterogeneous clinical phenotypes, such as the behavioral variant (bvFTD) in the affected men and the nonfluent/agrammatic variant of primary progressive aphasia (nfvPPA) in the affected woman. Haploinsufficiency was revealed by both quantitative real-time PCR of the gene and protein analyses. These findings provide further support for a previously proposed role for the GRN gene in the genetic etiology of FTD and its phenotypic variability.
Assuntos
Demência Frontotemporal/genética , Deleção de Genes , Predisposição Genética para Doença/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Idoso , Idoso de 80 Anos ou mais , Éxons/genética , Feminino , Genes Dominantes/genética , Estudos de Associação Genética , Haploinsuficiência/genética , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Progranulinas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.
Assuntos
Cromatina/metabolismo , DNA/metabolismo , Mytilus/metabolismo , Protaminas/metabolismo , Espermatozoides/metabolismo , Animais , Cromatina/genética , DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Histonas/metabolismo , Masculino , Nuclease do Micrococo/metabolismo , Mytilus/genética , Protaminas/isolamento & purificação , Ligação Proteica , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismoRESUMO
Sea urchin DNA (cytosine-5)-methyltransferase (Dnmt1) that is responsible for maintenance of DNA methylation patterns clearly shares similarity with various Dnmt1s identified in vertebrates. In this study, we determined the structure of the sea urchin Dnmt1 gene by screening a genomic library of the sea urchin Paracentrotus lividus with the complementary DNA (cDNA) as probe. Analysis of the positive clones demonstrated that the Dnmt1 gene consists of 34 exons and 33 introns spanning a distance of 35 kb. All exon-intron junction sequences agree with the GT/AG consensus with the exception of the 3' acceptor site of intron 8 where CT replaces AG consensus. The differences in the total number of exons between sea urchin and mouse genes reside mainly in the N-terminal region of the protein (exons 5-7 of the sea urchin, exons 5-12 of the mouse) where there is very low similarity in the amino acid sequence. By reverse transcription-polymerase chain reaction using oligonucleotides spanning different regions of the cDNA we carried out a comprehensive analysis of alternative splicing of the Dnmt1 messenger RNA (mRNA) in sea urchin embryos at different stages of development. We demonstrated the presence of at least five alternative spliced mRNAs that are regulated during development and are translated in truncated or deleted proteins.
Assuntos
Processamento Alternativo , DNA (Citosina-5-)-Metiltransferases/genética , Ouriços-do-Mar/genética , Animais , Sequência de Bases , Western Blotting , DNA/química , DNA/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Complementar/genética , Éxons , Genes/genética , Íntrons , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ouriços-do-Mar/enzimologia , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Methylation pattern has been studied in two genes of sea urchin Paracentrotus lividus using sodium bisulfite method to understand the possible role of DNA methylation during invertebrate development. Three regions of the gene for the hatching enzyme have been analyzed and all of them resulted unmethylated in embryos at different stages of development. Four CpG rich regions have been studied in the gene for DNA methyltransferase: upstream, upstream-exon1, intron 1 and exon 20. The upstream-exon 1 region is always unmethylated, while intron 1 and exon 20 are heavy methylated. Only the upstream fragment changed its pattern of methylation during development. For none of the studied regions the reported data show a general direct correlation between gene expression and methylation process during development.
Assuntos
Metilação de DNA , Paracentrotus/embriologia , Paracentrotus/genética , Animais , DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência de DNA , Sulfitos/metabolismoRESUMO
Supercoiled state corresponds to the active form for plasmid applications. The relaxed circular form of plasmids is often inactive or poorly active. To obtain significant amounts of almost fully supercoiled DNA, we modified the standard protocol of a commercially available Qiagen plasmid purification kit. Our changes led to isolation of almost 100% of the plasmids in the supercoiled state. The modified protocol was used to purify different plasmids with consistent results. The purified plasmids maintain supercoiled state for about two months. The modified protocol is very advantageous because it allows easy DNA production with high degree of supercoiled form at low cost.
Assuntos
DNA Bacteriano/isolamento & purificação , DNA Super-Helicoidal/isolamento & purificação , Escherichia coli/genética , Plasmídeos/isolamento & purificação , Cromatografia por Troca Iônica , Criopreservação , TemperaturaRESUMO
The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be â¼1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.
Assuntos
Proteínas Nucleares/química , Poliquetos/química , Homologia de Sequência de Aminoácidos , Espermatozoides/química , Sequência de Aminoácidos , Animais , Masculino , Dados de Sequência Molecular , Protaminas/química , Alinhamento de SequênciaRESUMO
Recently cortical spreading depression (CSD) has been hypothesized to involve epigenetic control of gene expression, by inducing an overall decrease of H3K4 and increase of H3K9 di-methylation. Here we evaluated the H3K4 and H3K9 di-methylation level at specific loci in rat brains 24 h after CSD induction. Analysis of two selected neuroprotective genes, iNOS and HIF-1α, showed marked increase in lysine 4 di-methylation and decrease in lysine 9 di-methylation of H3 histone. In addition, di-methylation of H3K4 increased moving toward 5' end of the genes in CSD-induced rat hemispheres. Such behavior may reflect an epigenetic molecular memory of actively transcribed genes. We extended our analysis on the H3K4 and H3K9 di-methylation levels of two long interspersed sequences (LINEs). We showed that CSD induction led to di-methylation decrease in lysine 4 and increase in lysine 9 of H3 histone, a trend which reflected the overall chromatin changes previously demonstrated. In conclusion, our data corroborate the hypothesis that epigenetic regulation of gene expression can be affected by CSD and that might be a pivotal molecular mechanism of CSD-induced preconditioning phenomenon which induces tolerance to a subsequent episode of ischemia. In such control, we evidenced two effects: i) a molecular memory of transcribed neuroprotective genes, ii) an epigenetic silencing of retrotransposable sequences.
Assuntos
Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Histonas/metabolismo , Lisina/metabolismo , Retroelementos/genética , Animais , Núcleo Celular/química , Centrifugação com Gradiente de Concentração , Hidrato de Cloral/farmacologia , Imunoprecipitação da Cromatina , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Fator 1 Induzível por Hipóxia/biossíntese , Fator 1 Induzível por Hipóxia/genética , Masculino , Metilação , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The mode of binding of sperm and somatic H1 histones to DNA has been investigated by analyzing the effect of their addition on the electrophoretic mobility of linear and circular plasmid molecules. Low concentrations of sperm histones do not appear to alter the electrophoretic mobility of DNA, whereas at increasing concentrations, an additional DNA band is observed near the migration origin. This band then becomes the only component at higher values. In contrast, somatic histones cause a gradual retardation in the mobility of the DNA band at low concentrations and aggregated structures are observed only at higher values. Experiments on the H1 globular domain obtained by limited proteolysis indicate that the mode of binding to DNA depends on the H1 globular domain. The arginine residues appear to be relevant for the different effects as indicated by experiments on sperm histone and on protamine with arginines deguanidinated to ornithines. The modified molecules influence DNA mobility like somatic H1s, indicating that the positive guanidino groups of arginines cannot be substituted by the positive amino groups of ornithines. Modifications of the amino groups of lysines show that these residues are necessary for the binding of H1 histones to DNA but they have no influence on the binding mode.
Assuntos
Arginina/metabolismo , DNA/metabolismo , Histonas/metabolismo , Arginina/genética , Fenômenos Biofísicos , DNA/genética , Histonas/química , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Masculino , Ornitina/genética , Ornitina/metabolismo , Plasmídeos , Protaminas/genética , Protaminas/metabolismo , Espermatozoides/metabolismoRESUMO
Obesity is associated with an increased risk of breast cancer. A number of adipocytokines are increased in obesity causing low-level chronic inflammation associated with an increased risk of tumors. The adipocytokine leptin shows profound anti-obesity and pro-inflammatory activities. We have hypothesized that in common obesity, high circulating leptin levels might contribute to an increased risk of breast cancer by affecting mammary cell proliferation and survival. Leptin exerts its activity not only through leptin receptor (LepR), but also through crosstalk with other signaling systems implicated in tumorigenesis. In this study, we focused our attention on the relationship between the leptin/LepR axis and the estrogen receptor-alpha (ERalpha). To this aim, we utilized two human breast cancer cell lines, one ERalpha-positive cell line (MCF 7) and the other ERalpha-negative cell line (MDA-MB 231). We observed that the two cell lines had a different sensitivity to recombinant leptin (rleptin): on MCF 7 cells, rleptin induced a strong phosphorylation of the signal transducer and activator of transcription (STAT) 3 and of the extracellular related kinase 1/2 pathways with an increased cell viability and proliferation associated with an increased expression of ERalpha receptor. This response was not present in the MDA-MB 231 cells. The effects induced by leptin were lost when LepR was neutralized using either a monoclonal inhibitory antibody to LepR or LepR gene-silencing siRNA. These data suggest that there is a bidirectional communication between LepR and ERalpha, and that neutralization and/or inactivation of LepR inhibits proliferation and viability of human breast cancer cell lines. This evidence was confirmed by ex vivo studies, in which we analyzed 33 patients with breast cancer at different stages of disease, and observed that there was a statistically significant correlation between the expression of LepR and ERalpha. In conclusion, this study suggests a crosstalk between LepR and ERalpha, and could envisage novel therapeutic settings aimed at targeting the LepR in breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptores para Leptina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Mama/efeitos dos fármacos , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Leptina/metabolismo , Leptina/farmacologia , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estadiamento de Neoplasias , Fosforilação/efeitos dos fármacos , Receptores para Leptina/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estatísticas não ParamétricasRESUMO
Preconditioning with Cortical Spreading Depression induces a sort of tolerance to a subsequent episode of ischemia. The mechanism of this tolerance is not clear. We studied if such treatment induces epigenetic chromatin modifications on the hemispheres of rats preconditioned by Cortical Spreading Depression. The contralateral hemispheres were used as control. We determined the level of H3K4 and H3K9 methylation and the mRNA amounts for the two well known H3K4 methyltransferases (MLL and SET7) in rats 24 degrees h after the Cortical Spreading Depression induction. Western blotting experiments have been performed using three different types of primary antibodies against mono-, di- and tri-methyl H3K4 and primary antibody anti-dimethyl H3K9. In the same samples we checked if the H3 histones were replaced by the H3.3 histone variants that could be an additional marker of chromatin modifications. The level of mono- and di-methyl H3K4 was significantly lower in samples of the treated hemispheres than those of the contralateral hemispheres (40% and about 60%, respectively) while the level of tri-methylation remained unchanged. The level of di-methyl H3K9 was almost 60% higher in the treated hemispheres than the contralateral hemispheres. The treatment for Cortical Spreading Depression affected also the level of expression of H3K4 histone methyltransferase MLL and SET7 that decreased in the treated hemispheres in comparison to the control hemispheres (80% and 40%, respectively). The treatment for Cortical Spreading Depression instead had no effects on the overall amounts of mRNA for H3 and H3.3 histones. In conclusion epigenetic chromatin modifications are evident in rats 24 degrees h after the Cortical Spreading Depression induction.
Assuntos
Córtex Cerebral/metabolismo , Cromatina/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/genética , Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Western Blotting , Córtex Cerebral/enzimologia , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histonas/classificação , Histonas/genética , Masculino , Metilação , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Methylation of specific lysine residues in histone tails has been proposed to function as a stable epigenetic marker that directs biological functions altering chromatin structure. Recent findings have implicated alteration in heterochromatin formation as a contributing factor in cancer development. In order to verify whether changes in the overall level of H3K4 histone methylation could be involved in oral squamous carcinoma, the levels of H3K4me1, me2 and me3 were measured in oral squamous carcinoma, leukoplakias and normal tissues. The levels of H3K4me2 and me3 were significantly different in oral squamous cell carcinoma in comparison with normal tissue: the level of H3K4me2 was increased while that of H3K4me3 decreased. No significant differences could be found between the two types of tissues in the level of H3K4me1. A similar trend was found in the leukoplakias that appeared more like the pathological than normal tissue. These results support the idea that alteration of chromatin structure could contribute to oncogenic potential.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Histonas/metabolismo , Neoplasias Bucais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Feminino , Humanos , Técnicas In Vitro , Leucoplasia Oral/metabolismo , Masculino , Metilação , Pessoa de Meia-IdadeRESUMO
Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-L-lysine (poly-lys), α-poly-L-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-L-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-L-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.
RESUMO
The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.