RESUMO
Cytosine DNA methylation is a significant form of DNA modification closely associated with gene expression in eukaryotes, fungi, animals, and plants. Although the reference genomes of cotton (Gossypium hirsutum L.) have been publically available, the salinity-stress-induced DNA methylome alterations in cotton are not well understood. Here, we constructed a map of genome-wide DNA methylation characteristics of cotton leaves under salt stress using the methylated DNA immunoprecipitation sequencing method. The results showed that the methylation reads on chromosome 9 were most comparable with those on the other chromosomes, but the greatest changes occurred on chromosome 8 under salt stress. The DNA methylation pattern analysis indicated that a relatively higher methylation density was found in the upstream2k and downstream2k elements of the CDS region and CG-islands. Almost 94% of the reads belonged to LTR-gspsy and LTR-copia, and the number of methylation reads in LTR-gypsy was four times greater than that in LTR-copia in both control and stressed samples. The analysis of differentially methylated regions (DMRs) showed that the gene elements upstream2k, intron, and downstream2k were hypomethylated, but the CDS regions were hypermethylated. The GO (Gene Ontology) analysis suggested that the methylated genes were most enriched in cellular processes, metabolic processes, cell parts and catalytic activities, which might be closely correlated with response to NaCl stress. In this study, we completed a genomic DNA methylation profile and conducted a DMR analysis under salt stress, which provided valuable information for the better understanding of epigenetics in response to salt stress in cotton.
Assuntos
Metilação de DNA , Genoma de Planta , Gossypium/genética , Salinidade , Estresse Fisiológico , Cromossomos de Plantas/genética , Estudo de Associação Genômica AmplaRESUMO
Phospholipase Cg2 (PLCg2) induces apoptosis of immune and tumor cells; however, it remains unclear whether PLCg2 promotes hepatocyte apoptosis during liver regeneration (LR). Therefore, to establish a framework for further exploring the function of PLCg2, we generated recombinant adenoviruses carrying a template encoding short hairpin (sh)-RNA targeting PLCg2 (Ad-PLCg2-shRNA), which were used to silence the expression of PLCg2 in BRL-3A cells. First, three pairs of PLCg2-shRNAs were designed, synthesized, and cloned into a shuttle vector, pHBAd-U6-GFP, after annealing. The recombinant shuttle plasmids were co-transfected with the backbone vector pHBAd-BHG into HK293 cells to package the recombinant Ad-PLCg2-shRNAs used to infect BRL-3A cells. Infection efficiency was monitored by observing the number of GFP-positive cells under a fluorescent microscope. To determine the recombinant adenoviruses with the highest silencing efficiency, levels of PLCg2 mRNA were evaluated by qRT-PCR. DNA sequencing confirmed that the correct shRNA coding sequences were inserted into the shuttle vectors and adenoviral plasmids. The titers of three recombinant adenoviruses were at least 1 x 10(10) PFU/mL. The most effective adenoviral construct, with interference efficiency of 77%, was determined by qRT-PCR. These results show that a recombinant adenovirus, Ad-PLCg2-shRNA, was developed and was effective at silencing the rat PLCg2 gene. This construct may contribute to the study of PLCg2 in hepatocyte apoptosis during LR.
Assuntos
Adenoviridae/genética , Hepatócitos/metabolismo , Regeneração Hepática/genética , Fosfolipase C gama/genética , Animais , Apoptose/genética , Linhagem Celular , Vetores Genéticos/genética , Fosfolipase C gama/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , TransfecçãoRESUMO
Isoflurane can induce widespread cytotoxicity. We hypothesized that isoflurane induces apoptosis partly by causing excessive calcium release from the endoplasmic reticulum (ER) via direct activation of inositol 1,4,5-trisphosphate receptors (IP3R). Rat pheochromocytoma cells cultured for seven days with nerve growth factor were divided into four groups: control group (C), IP3R antagonist group (X), isoflurane group (I) and isoflurane + IP3R antagonist group (I+X). Groups I and I+X were treated with 1 MAC isoflurane for 12 h. Groups X and I+X were pretreated with IP3R antagonist. Annexin V/PI apoptosis and TUNEL assays were performed to evaluate cell apoptosis. TEM was used to observe changes in cell ultrastructure. Changes in calcium concentration ([Ca(2+)]i) in the cytoplasm were measured by flow cytometry. RT-PCR was performed to evaluate IP3R mRNA expression. TEM showed that isoflurane treatment altered cell ultrastructure. Compared to group C, cell apoptosis rate and [Ca(2+)]i increased in groups I and I+X (P < 0.05). Compared to group C, IP3R mRNA expression was lower in group X and higher in group I (P < 0.05). Compared to group X, cell apoptosis rate, [Ca(2+)]i and IP3R mRNA expression increased in groups I and I+X (P < 0.05). Compared to group I, cell apoptosis rate, [Ca(2+)]i and IP3R mRNA expression decreased in group I+X (P < 0.05). These results suggest that exposure to 1 MAC isoflurane for 12 h causes excessive calcium release partly by direct activation of IP3R on the ER membrane and triggers cell apoptosis.
Assuntos
Anestésicos Inalatórios/toxicidade , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Isoflurano/toxicidade , RNA Mensageiro/metabolismo , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Receptores de Inositol 1,4,5-Trifosfato/agonistas , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Transporte de Íons , Compostos Macrocíclicos/farmacologia , Fator de Crescimento Neural/farmacologia , Oxazóis/farmacologia , Células PC12 , RNA Mensageiro/agonistas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RatosRESUMO
Hemangioblastoma of the central nervous system occurs as sporadic tumors or as a part of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary tumor syndrome caused by a germline mutation in the VHL tumor suppressor gene. We screened a Chinese family with VHL for mutations in the VHL gene and evaluated a genetic test for diagnosing VHL disease and clinical screening of family members. DNA extracted from the peripheral blood of all live members and from tissue of deceased family members with VHL disease was amplified by polymerase chain reaction to 3 VHL gene exons. Mutations in the amplification products were compared against the Human Gene Mutation Database. The involvement of multiple organs among the kindred with VHL disease was confirmed by medical history and radiography. Of the 12 members of the 4-generation family, 5 were diagnosed with VHL disease. Patient age at the initial diagnosis was 26-36 years (mean = 31 years). The mean time was 15 (11-19 months) from symptom appearance to the first patient visit to the hospital. Sequence analysis revealed that the frameshift mutation 327del C (p.Gly39Alafs*26) in exon 1 affected all family members, but not the healthy individuals or 16 unrelated controls. Members without gene mutation showed no clinical manifestation of VHL disease. We detected a conserved novel frameshift mutation in the VHL gene of the family members that contributes to VHL. DNA analysis of VHL is advantageous for VHL diagnosis. We developed a quick and reliable method for VHL diagnosis.
Assuntos
Mutação da Fase de Leitura , Hemangioblastoma/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Adulto , Análise Mutacional de DNA , Feminino , Testes Genéticos , Hemangioblastoma/diagnóstico , Hemangioblastoma/etiologia , Humanos , Masculino , Linhagem , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/diagnósticoRESUMO
Colony stimulating factors (CSF) have been considered to modulate liver regeneration (LR) after partial hepatectomy (PH) at the tissue level. However, it remains unclear about precise mechanism of action of CSF in regeneration at the cellular level. Therefore, eight rat liver cell types were isolated by Percoll gradient centrifugation and magnetic beads. CSF-mediated signaling pathway genes were obtained by searching the related pathway databases and their expression profiles in 8 hepatic cell types were measured using rat Genome 230 2.0 Microarray. RT-PCR was performed to assess the reliability of chip results. The result showed a large difference in expression profiles of CSF-mediated signaling pathway genes between different cell types; most genes involved in CSF-mediated signaling pathways were mainly unregulated across liver cell samples. The implication of these genes in LR was analyzed by the bioinformatics and systems biology method. According to chip results and gene synergy, a significant enhancement of the CSF3-mediated Pi3k/Akt pathway at 30-36 h in hepatocytes and at 24 h in biliary epithelial cells post-PH could be associated with active proliferation in these two cell types; the striking decrease in Jak/Stat cascade activity in hepatic stellate cells at 2 and 12 h post- PH or even inactive in dendritric cells during the whole LR implied that proliferation of these two cell types is possibly regulated by other signaling pathways. These data suggest the potential relevance of CSF in liver regeneration at the cellular level.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Regeneração Hepática/genética , Fígado/metabolismo , Animais , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Hepatectomia , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Janus Quinases/genética , Janus Quinases/metabolismo , Células de Kupffer/citologia , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Fígado/cirurgia , Análise em Microsséries , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de SinaisRESUMO
It has been well established that ERK1/2 signaling, often subdivided into nine types of pathways, can regulate the hepatocyte proliferative response during liver regeneration. However, the effect of ERK1/2 signaling on the proliferation of other hepatic cell types remains unclear. We isolated and purified 8 liver cell types at 10 time points after 2/3 hepatectomy in adult rats. For each cell type, mRNA expression changes for ERK1/2 signaling-involved genes were monitored up to 168 h, using microarrays. Real-time PCR assays were performed for array data verification. The expression levels of these genes varied considerably between different cell types. Integrating microarray results with gene synergical analysis, at the priming phase, activation of integrin/Grb2/Ras pathway in hepatocytes apparently contributed to G0/G1 transition. Two other pathways, G-protein/EPAC/Rap1 and G-protein/PKA/Rap1, were stimulated in hepatic stellate cells, while RTK/PKC/Ras and RTK/Grb2/Ras were stimulated in Kupffer cells. At the progressive phase, the ERK1/2 pathway is involved in hepatocyte replication; three pathways, namely Ca(2+)/PKC/Ras, RTK/Grb2/Ras and G-protein/EPAC/Rap1, were found to play roles in biliary epithelial cell proliferation, while RTK/PKC/Ras and RTK/Grb2/Ras were involved in Kupffer cell proliferation, and G-protein/PKC/Ras in pit cell proliferation. At the terminal phase, the promotive effect of the ERK1/2 pathway on replication of hepatocytes, biliary epithelial cells, oval cells, hepatic stellate cells, Kupffer cells, and dendritic cells was considerably reduced, possibly due to their differentiation at the end of regeneration. G-protein/PKC/Ras, integrin/Grb2/Ras and G-protein/ PKA/Rap1 pathways were active in sinusoidal endothelial cells, perhaps to aid in their proliferation. We conclude that ERK1/2 has a signaling role in the regulation of proliferation of 8 cell types during liver regeneration process.
Assuntos
Proliferação de Células , Regeneração Hepática/genética , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Animais , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Fígado/citologia , Fígado/cirurgia , Masculino , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hepatic pit cells are a population of large granular lymphocytes that substantially contribute to hepatic immunity. Studies have proven that pit cells have a role in liver regeneration, but the details of the relationship between pit cells and liver regeneration is not clear at present. We subjected rats to a two-third hepatectomy; pit cells with high purity were obtained with Percoll density centrifugation and immunomagnetic bead methods, and the changes in mRNA levels in pit cells from the regenerating liver were monitored up to 168 h using a Rat Genome 230 2.0 Array composed of 25,020 distinct rat liver cDNA clones. Of the 25,020 genes analyzed, 612 known and 358 unknown genes were identified to be associated with liver regeneration. The 612 known genes are classified into up-regulation and down-regulation patterns based on the expression levels; they primarily participate in at least 23 biological activities based on gene ontology analysis. Together with gene function enrichment analysis, cytokines and a growth factor-mediated pathway in pit cells were activated at an early phase of liver regeneration; pit cell proliferation occurred from 24-72 h after liver hepatectomy; the machinery of pit cell differentiation commenced early and came into play late; an immune/inflammatory response was enhanced late. Expression pattern analysis of functionally classified genes in pit cells can give insights into the relationship between pit cells and liver regeneration.
Assuntos
Regulação da Expressão Gênica , Células Matadoras Naturais , Regeneração Hepática , Fígado/fisiologia , Animais , DNA Complementar , Perfilação da Expressão Gênica , Hepatectomia , Imunidade Inata , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Fígado/citologia , Análise em Microsséries , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.