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1.

Objective

To identify reliable reference genes for gene expression analysis in Gossypium raimondii.

Results

Five different software tools, geNorm, NormFinder, BestKeeper, ReFinder and ?Ct method were employed to analyze the qRT-PCR data systematically of 12 housekeeping genes. SAD and TUA11 showed relatively stable expression levels in all tissues (i.e. leaves, shoots, buds, and sepals). We then limited our analysis to each plant part and identified tissue-specific reference genes. Our results showed TUA11, TUB6 and EF1a, EF1a, MZA and GAPC2, MZA, GAPC2, SAD and TUA11, and UBQ and MZA were reliable reference genes in leaves, shoots, buds, and sepals, respectively.

Conclusion

Some genes were commonly identified as candidate reference genes in more than two tissue, while others were tissue-specific. Thus, our study allows choosing an appropriate control gene based on sampling for gene expression analysis.
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2.

Background

The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII) including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26) and their receptors (HCRII). Despite the report of a near complete pufferfish (Takifugu rubripes) genome sequence, these genes remain undescribed in fish.

Results

We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF). Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes.

Conclusion

We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish.
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3.

Background

We previously demonstrated that the local immune status correlated with the glioma prognosis. Interleukin-6 (IL6) was identified as an important local immune-related risk marker related to unfavourable prognosis. In this study, we further investigated the role and regulation of IL6 signalling in glioma.

Methods

The expression and prognostic value of IL6 and the IL6 receptor (IL6R) were explored in The Cancer Genome Atlas (TCGA) and REMBRANDT databases and clinical samples. Functional effects of genetic knockdown and overexpression of IL6R or IL6 stimulation were examined in vitro and in tumours in vivo. The effects of the nuclear factor of activated T cells-1 (NFAT1) on the promoter activities of IL6R and IL6 were also examined.

Results

High IL6- and IL6R-expression were significantly associated with mesenchymal subtype and IDH-wildtype gliomas, and were predictors of poor survival. Knockdown of IL6R decreased cell proliferation, invasion and neurosphere formation in vitro, and inhibited tumorigenesis in vivo. IL6R overexpression or IL6 stimulation enhanced the invasion and growth of glioma cells. TCGA database searching revealed that IL6- and IL6R-expression were correlated with that of NFAT1. In glioma cells, NFAT1 enhanced the promoter activities of IL6R and IL6, and upregulated the expression of both IL6R and IL6.

Conclusion

NFAT1-regulated IL6 signalling contributes to aggressive phenotypes of gliomas, emphasizing the role of immunomodulatory factors in glioma malignant progression.
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4.

Background

The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits.

Results

We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2.

Conclusions

Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.
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5.
6.
7.

Background

Recent studies on the involvement of the G12 family of heterotrimeric G proteins (Gα12 and Gα13, the products of the GNA12 and GNA13 genes, respectively) in oncogenic pathways have uncovered a link between G12 signaling and cancer progression. However, despite a well characterized role of Rho GTPases, the potential role of secreted factors in the capacity of G12 signaling to promote invasion of cancer cells is just beginning to be addressed.

Methods

MDA-MB-231 and MCF10A breast cancer cell lines were employed as a model system to explore the involvement of secreted factors in G12-stimulated cell invasion. Factors secreted by cells expressing dominant-active Gα12 were identified by protein array, and their involvement in breast cancer cell invasion was assessed through both RNAi-mediated knockdown and antibody neutralization approaches. Bioinformatics analysis of the promoter elements of the identified factors suggested NF-κB elements played a role in their enhanced expression, which was tested by chromatin immunoprecipitation.

Results

We found that signaling through the Gα12 in MDA-MB-231 and MCF10A breast cancer cell lines enhances expression of interleukins (IL)-6 and ?8, and matrix metalloproteinase (MMP)-2, and that these secreted factors play a role in G12-stimulated cell invasion. Furthermore, the enhanced expression of these secreted factors was found to be facilitated by the activation of their corresponding promoters, where NF-κB seems to be one of the major regulators. Inhibition of IL-6 and IL-8, or MMP-2 activity significantly decreased Gα12-mediated cell invasion.

Conclusions

These studies confirm and extend findings that secreted factors contribute to the oncogenic potential of G12 signaling, and suggest potential therapeutic targets to control this process.
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8.
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10.

Background

Implantation of the embryo and successful pregnancy are dependent on the differentiation of endometrial stromal cells into decidual cells. Female interleukin-11 receptor α (IL-11Rα) deficient mice are infertile due to disrupted decidualization, suggesting a critical role for IL-11 and its target genes in implantation. The molecular targets of IL-11 in the uterus are unknown, but it is likely that IL-11 signaling modifies the expression of other genes important in decidualization. This study aimed to identify genes regulated by IL-11 during decidualization in mouse uterus, and to examine their expression and localization as an indication of functional significance during early pregnancy.

Methods

Decidualization was artificially induced in pseudopregnant wild type (IL11Ra+/+) and IL-11Rα deficient (IL11Ra-/-) littermates by oil injection into the uterine lumen, and gene expression analyzed by NIA 15K cDNA microarray analysis at subsequent time points. Quantitative real-time RT-PCR was used as an alternative mRNA quantitation method and the expression and cellular localization of the protein products was examined by immunohistochemistry.

Results

Among 15,247 DNA probes, 13 showed increased and 4 decreased expression in IL11Ra-/- uterus at 48 h of decidualization. These included 4 genes encoding extracellular matrix proteins; collagen III α1, secreted acidic cysteine-rich glycoprotein (SPARC), biglycan and nidogen-1 (entactin). Immunohistochemistry confirmed increased collagen III and biglycan protein expression in IL11Ra-/- uterus at this time. In both IL11Ra-/- and wild type uterus, collagen III and biglycan were primarily localized to the outer connective tissue and smooth muscle cells of the myometrium, with diffuse staining in the cytoplasm of decidualized stromal cells.

Conclusion

These data suggest that IL-11 regulates changes in the uterine extracellular matrix that are necessary for decidualization.
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11.

Objective

Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that has critical roles in the treatment of atherosclerosis, hyperlipidemia and hepatic steatosis.

Results

Squalene is a novel nature PPARα agonist, identified from reporter gene assay and qPCR analysis. Cultured hepatocytes stimulated with squalene exhibited significantly decreased cellular triacylglycerols and cholesterol concentrations, while cellular uptake of fatty acids was increased. Quantitative PCR analysis revealed that expression of genes related to fatty acid uptake, fatty acid oxidation, ketogenesis and reverse cholesterol transport metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed in cell treated with squalene.

Conclusion

Squalene is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that regulates the expression of lipid metabolism genes in hepatocytes.
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12.

Background

Differential gene expression is important to understand the biological differences between healthy and diseased states. Two common sources of differential gene expression data are microarray studies and the biomedical literature.

Methods

With the aid of text mining and gene expression analysis we have examined the comparative properties of these two sources of differential gene expression data.

Results

The literature shows a preference for reporting genes associated to higher fold changes in microarray data, rather than genes that are simply significantly differentially expressed. Thus, the resemblance between the literature and microarray data increases when the fold-change threshold for microarray data is increased. Moreover, the literature has a reporting preference for differentially expressed genes that (1) are overexpressed rather than underexpressed; (2) are overexpressed in multiple diseases; and (3) are popular in the biomedical literature at large. Additionally, the degree to which diseases are similar depends on whether microarray data or the literature is used to compare them. Finally, vaguely-qualified reports of differential expression magnitudes in the literature have only small correlation with microarray fold-change data.

Conclusions

Reporting biases of differential gene expression in the literature can be affecting our appreciation of disease biology and of the degree of similarity that actually exists between different diseases.
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13.

Background

One of the microorganisms from dental plaque associated with severe inflammatory responses in infectious endocarditis is Porphyromonas gingivalis. It is a Gram-negative bacteria harvested from chronic periodontitis patients. Lipopolysaccharide (LPS) obtained from P. gingivalis promotes the expressions of interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-α). Flavonoids are thought to participate in processes that control inflammation, such as the expression of cyclooxygenase-2 (COX-2).

Methods

We investigated the effects of luteolin, quercetin, genistein and quercetagetin on cardiomyoblasts treated with LPS alone or in combination with following inhibitors p38 (SB203580), ERK (PD98059), JNK (SP600125) and PKC (Calphostin C) for 1 h. The kinase activation and COX-2 expression levels were determined at the gene and protein levels.

Results

These flavonoids are considered to inhibit the activation of mitogen-activated protein kinase (MAPK) and the degradation of inhibitor of kappa B-alpha (IκB-α). They also play a role in COX-2 expression.

Conclusion

We conclude that the tested flavonoids inhibit inflammatory responses induced by LPS in H9c2 cells.
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14.
15.
16.

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.
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17.

Background

INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer.

Results

We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine IL-8 and induced PAK6. These genes were regulated in a reciprocal manner following downregulation of INPP4B in the independently derived INPP4B-positive LNCaP prostate cancer cell line. Inhibition of PI3K/Akt pathway, which is highly active in both PC-3 and LNCaP cells, did not reproduce INPP4B mediated suppression of IL-8 mRNA expression in either cell type. In contrast, inhibition of PKC signaling phenocopied INPP4B-mediated inhibitory effect on IL-8 in either prostate cancer cell line. In PC-3 cells, INPP4B overexpression caused a decline in the level of metastases associated BIRC5 protein, phosphorylation of PKC, and expression of the common PKC and IL-8 downstream target, COX-2. Reciprocally, COX-2 expression was increased in LNCaP cells following depletion of endogenous INPP4B.

Conclusion

Taken together, we discovered that INPP4B is a novel suppressor of oncogenic PKC signaling, further emphasizing the role of INPP4B in maintaining normal physiology of the prostate epithelium and suppressing metastatic potential of prostate tumors.
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18.

Background

Stress is a recognized risk factor for mood and anxiety disorders that occur more often in women than men. Prefrontal brain regions mediate stress coping, cognitive control, and emotion. Here, we investigate sex differences and stress effects on prefrontal cortical profiles of gene expression in squirrel monkey adults.

Methods

Dorsolateral, ventrolateral, and ventromedial prefrontal cortical regions from 18 females and 12 males were collected after stress or no-stress treatment conditions. Gene expression profiles were acquired using HumanHT-12v4.0 Expression BeadChip arrays adapted for squirrel monkeys.

Results

Extensive variation between prefrontal cortical regions was discerned in the expression of numerous autosomal and sex chromosome genes. Robust sex differences were also identified across prefrontal cortical regions in the expression of mostly autosomal genes. Genes with increased expression in females compared to males were overrepresented in mitogen-activated protein kinase and neurotrophin signaling pathways. Many fewer genes with increased expression in males compared to females were discerned, and no molecular pathways were identified. Effect sizes for sex differences were greater in stress compared to no-stress conditions for ventromedial and ventrolateral prefrontal cortical regions but not dorsolateral prefrontal cortex.

Conclusions

Stress amplifies sex differences in gene expression profiles for prefrontal cortical regions involved in stress coping and emotion regulation. Results suggest molecular targets for new treatments of stress disorders in human mental health.
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19.

Background

The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos.

Results

Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.

Conclusion

These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.
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20.

Introduction

Gentian spotted bleaching disease (GSBD), a novel disease of unknown etiology, affects Gentiana triflora plants that are cultivated as ornamental flowers in Japan. This disease leads to the production of necrotic leaf spots, a delay in flowering, and has thus become a serious problem for gentian production.

Objectives

The objective of this study was to identify the cause of GSBD in G. triflora by analyzing differences between healthy and GSBD-affected leaves.

Method

Selected metabolite concentrations in healthy and GSBD-affected leaves were quantified using capillary electrophoresis and liquid chromatography-mass spectrometry, and statistically significant differences in metabolite concentrations were assessed. GSBD-affected metabolic pathways were identified followed by examination of pathway-related gene expression and enzyme activities. Furthermore, the effects of root hypoxia on metabolite concentrations and gene expression were investigated.

Results

We found that concentrations of Calvin cycle intermediates and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity were significantly lower in GSBD-affected leaves, whereas sucrose cleavage and Ala accumulation were enhanced. Since these metabolic changes are frequently observed in plants exposed to hypoxia, the expression of hypoxia-responsive genes was investigated. Expression levels of hypoxia-responsive genes were higher in GSBD-affected plants than in the controls. Furthermore, root hypoxia induced similar symptoms and metabolic changes as those observed in GSBD-affected plants.

Conclusion

Our results indicate that GSBD was likely induced by root hypoxia and that metabolome analysis is an effective tool for identifying the cause of plant disease with unknown etiologies.
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