Project description:We used quantitative mass spectrometry-based proteomics to unravel global nerve growth factor (NGF)-induced changes in protein abundance using the rat PC12 cell line. Cells were stimulated with NGF for 0, 24 and 48 h in a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10). All experiments were performed as biological replicates.
Project description:A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver. This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection. The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific. Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver. 18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections. Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression. For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008)
Project description:Inflammation is a key component of pathological angiogenesis. Here we induce cornea neovascularisation using sutures placed into the cornea, and sutures are removed to induce a regression phase. We used whole transcriptome microarray to monitor gene expression profies of several genes
Project description:Spermatogenesis requires the presence of functional somatic Sertoli cells in the seminiferous tubules of the testis. Sertoli cells provide support and factors necessary for the successful progression of germ cells into spermatozoa. Sertoli cells are regulated to a large degree by the glycoprotein hormone FSH, which is required for the testis to acquire full size and spermatogenic capacity. Signaling events initiated by the binding of FSH to its receptor lead to an alteration of Sertoli cell gene expression. To characterize the changes in gene expression in FSH-treated Sertoli cells, we used the mRNA from these cells to screen Affymetrix U34A rat GeneChip oligonucleotide microarrays. Sertoli cells from 20-d-old rats were cultured in the presence of 25 ng/ml ovine FSH. At 0, 2, 4, 8, and 24 h after the addition of FSH, total RNA was purified and used to prepare biotinylated target, which was hybridized to the U34A rat microarray containing approximately 9000 rat genes. Analysis identified 100-300 transcripts at each time point that were up-regulated or down-regulated by 2-fold or greater. Genes previously reported to be FSH or cAMP regulated in rat Sertoli cells were identified, in addition to numerous genes not reported to be expressed or FSH regulated in Sertoli cells. The expression patterns of five of these genes, encoding nerve growth factor inducible gene B, PRL-1, PC3 nerve growth factor-inducible antiproliferative putative secreted protein, diacylglycerol acyltransferase, and an expressed sequence tag, in FSH- and N,O'-dibutyryl cAMP-treated rat Sertoli cells were confirmed and characterized by Northern blot analysis. Thus, we have begun to define the transcriptome induced and repressed by FSH in rat Sertoli cells, and we have generated datasets of genes available for further analysis in regard to spermatogenesis and Sertoli cell signaling.
Project description:We analyzed the comprehensive gene expression changes by nerve growth factor (NGF) in PC12 cells using Agilent microarrays. The genes of 1920 NGF treated were up-regulated and 2670 genes were down-regulated significantly compared with the control group. The expression level of genes involved in cell structure and proteolysis were up-regulated. On the other hand, the genes of cell cycle and some metabolism were rather down-regulated. Therefore, it is suggested that it is necessary to stop the cell cycle and down-regulate metabolism to neurite outgrowth. Gene regulation associated with the differentiation program in PC12 cells still needs to be elucidated. We analyzed the genes expression with Agilent microarrays after 6 hours of NGF treatment by the one-color method, and found 2099 genes were up-regulated and 2781 genes were down-regulated.
Project description:We analyzed the comprehensive gene expression changes by nerve growth factor (NGF) in PC12 cells using Agilent microarrays. The genes of 1920 NGF treated were up-regulated and 2670 genes were down-regulated significantly compared with the control group. The expression level of genes involved in cell structure and proteolysis were up-regulated. On the other hand, the genes of cell cycle and some metabolism were rather down-regulated. Therefore, it is suggested that it is necessary to stop the cell cycle and down-regulate metabolism to neurite outgrowth.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiae into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNAM-cM-^@M-^@microarray analysis using total RNAs harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis confirmed increased expression of miR-221, miR-181a* and miR-326, and decreased expression of miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Overexpression of miR-221 induced neurite outgrowth of PC12 cells in the absence of NGF treatment, and also enhanced neurite outgrowth caused by low-dose NGF. More importantly, knockdown of miR-221 by antagomir attenuated NGF-mediated neurite outgrowth. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are involved in apoptosis in PC12 cells. Our results indicate that miR-221 plays a critical role for neuronal differentiation as well as protection against apoptosis in PC12 cells. NGF induced miRNAs expression in rat pheochromocytoma PC12 cells was measured in cells treated with 100 ng/ml NGF for 0, 12, 24 and 48 hr.
Project description:Few studies have assessed the patterns of parasite populations of rodents over a longitudinal gradient in Chile. In this work, the gastrointestinal helminthic fauna of invasive rodents in Chile was examined to assess the association between their presence/absence and abundance with latitude, host sex, and host body condition, and to assess the coexistence and correlation of the abundance between parasite species. Rodents were obtained from 20 localities between 33 and 43°S. Helminths were extracted from the gastrointestinal tract and identified morphologically. Overall, 13 helminth taxa were obtained. The most frequently identified parasite species was Heterakis spumosa, and the most abundant was Syphacia muris, while Physaloptera sp. was the most widely distributed. No locality presented with a coexistence that was different from that expected by chance, while the abundance of five helminthic species correlated with the abundance of another in at least one locality, most likely due to co-infection rather than interaction. Host sex was associated with parasite presence or abundance, and female sex-biased parasitism was notably observed in all cases. Body condition and latitude presented either a positive or negative association with the presence or abundance of parasites depending on the species. It is notable that the likely native Physaloptera sp. is widely distributed among invasive rodents. Further, gravid females were found, suggesting spillback of this species to the native fauna. The low frequency and abundance of highly zoonotic hymenolepid species suggest that rodents are of low concern regarding gastrointestinal zoonotic helminths.