A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

The global effect of follicle-stimulating hormone and tumour necrosis factor α on gene expression in cultured bovine ovarian granulosa cells

Background Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Results Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and β1 and interleukin 1β. Conclusions In vitro, the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNFα. The response to TNFα indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNFα that displays features similar to immune cells.

Comparative effect of dairy fatty acids on cell adhesion molecules, nitric oxide and relative gene expression in healthy and diabetic human aortic endothelial cells

Dairy intake, despite its high saturated fatty acid (SFA) content, is associated with a lower risk of cardiovascular disease and diabetes. This in vitro study determined the effect of individual fatty acids (FA) found in dairy, and FA mixtures representative of a high SFA and a low SFA dairy lipid on markers of endothelial function in healthy and type II diabetic aortic endothelial cells.

Ascl1 coordinately regulates gene expression and the chromatin landscape during neurogenesis

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

Role of FTO in adipocyte development and function: recent insights

In 2007, FTO was identified as the first genome-wide association study (GWAS) gene associated with obesity in humans. Since then, various animal models have served to establish the mechanistic basis behind this association. Many earlier studies focussed on FTO’s effects on food intake via central mechanisms. Emerging evidence, however, implicates adipose tissue development and function in the causal relationship between perturbations in FTO expression and obesity. The purpose of this mini review is to shed light on these new studies of FTO function in adipose tissue and present a clearer picture of its impact on obesity susceptibility.

Regulation of gene and protein expression in cardiac myocyte hypertrophy and apoptosis

Considerable efforts have been expended in elucidating the inter-cellular and intra-cellular signaling pathways which elicit cardiac myocyte hypertrophy or apoptosis, and in identifying the changes which are associated with the end-stage of the response. The challenge now is to link the two. Although some of the signaling effects will be the acute modulation of existing protein function, long-term effects which bring about and maintain the hypertrophic state or which culminate in cell death are mediated at the level of gene and protein expression. With the advances in micro-array technology and genome sequencing, it is now possible to obtain a picture of the global gene expression profile in myocytes or in whole heart which dictates the proteins which could be made. This is not the final picture since additional regulation at the level of translation modulates the relative proportions of each protein that can be made from the transcriptome. Even here, further regulation of protein stability and turnover means that ultimately it is still necessary to examine the proteome to determine what may cause the functional changes in a cell. Thus, in order to gain a full picture of events which regulate the response and gain some insight into possible points of intervention for therapy, it is necessary to examine gene expression, mRNA translation and protein expression in concert.

Soil bacterial phoD gene abundance and expression in response to applied phosphorus and long-term management

Bacterial transformation of phosphorus (P) compounds in soil is largely dependent on soil microbial community function, and is therefore sensitive to anthropogenic disturbances such as fertilization or cropping systems. However, the effect of soil management on the transcription of bacterial genes that encode phosphatases, such as phoD, is largely unknown. This greenhouse study examined the effect of long-term management and P amendment on potential alkaline phosphatase (ALP) activity and phoD gene (DNA) and transcript (RNA) abundance. Soil samples (0–15 cm) were collected from the Glenlea Long-term Rotation near Winnipeg, Manitoba, to compare organic, conventional and prairie management systems. In the greenhouse, pots of soil from each management system were amended with P as either soluble mineral fertilizer or cattle manure and then planted with Italian ryegrass (Lolium multiforum). Soils from each pot were sampled for analysis immediately and after 30 and 106 days. Significant differences among the soil/P treatments were detected for inorganic P, but not the organic P in NaHCO3-extracts. At day 0, ALP activity was similar among the soil/P treatments, but was higher after 30 days for all P amendments in soil from organically managed plots. In contrast, ALP activity in soils under conventional and prairie management responded to increasing rates of manure only, with significant effects from medium and high manure application rates at 30 and 106 days. Differences in ALP activity at 30 days corresponded to the abundance of bacterial phoD genes, which were also significantly higher in soils under organic management. However, this correlation was not significant for transcript abundance. Next-generation sequencing allowed the identification of 199 unique phoD operational taxonomic units (OTUs) from the metagenome (soil DNA) and 35 unique OTUs from the metatranscriptome (soil RNA), indicating that a subset of phoD genes was being transcribed in all soils.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midguit cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3. (C) 2009 Elsevier Ltd. All rights reserved.

Dietary Free Oleic and Linoleic Acid Enhances Neutrophil Function and Modulates the Inflammatory Response in Rats

The high ingestion of oleic (OLA) and linoleic (LNA) acids by Western populations, the presence of inflammatory diseases in these populations, and the importance of neutrophils in the inflammatory process led us to investigate the effects of oral ingestion of unesterified OLA and LNA on rat neutrophil function. Pure OLA and LNA were administered by gavage over 10 days. The doses used (0.11, 0.22 and 0.44 g/kg of body weight) were based on the Western consumption of OLA and LNA. Neither fatty acid affected food, calorie or water intake. The fatty acids were not toxic to neutrophils as evaluated by cytometry using propidium iodide (membrane integrity and DNA fragmentation). Neutrophil migration in response to intraperitoneal injection of glycogen and in the air pouch assay, was elevated after administration of either OLA or LNA. This effect was associated with enhancement of rolling and increased release of the chemokine CINC-2 alpha beta. Both fatty acids elevated l-selectin expression, whereas no effect on beta(2)-integrin expression was observed, as evaluated by flow cytometry. LNA increased the production of proinflammatory cytokines (IL-1 beta and CINC-2 alpha beta) by neutrophils after 4 h in culture and both fatty acids decreased the release of the same cytokines after 18 h. In conclusion, OLA and LNA modulate several functions of neutrophils and can influence the inflammatory process.

Effect of medium/omega-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

Lipid emulsion (LE) containing medium/omega-6 long chain triglyceride-based emulsion (MCT/omega-6 LCT LE) has been recommended in the place of omega-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/omega-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/omega-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/omega-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/omega-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff - Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10 (- 3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p <= 0.001). We also showed that iodide excess inhibits the expression of essential genes for thyroid differentiation: Tshr, Nis, Tg, and Tpo. Relative expression of 14 of 20 transcripts selected by SAGE was confirmed by real-time PCR. Considering the key role of iodide organification in thyroid physiology, we also observed that both the oxidized form of iodide and iodide per se are responsible for gene expression modulation in response to iodide excess. (c) 2008 Elsevier Inc. All rights reserved.

Improved renal function after kidney transplantation is associated with heme oxygenase-1 polymorphism

Heme oxygenase-1 (HO-1) has a microsatellite polymorphism based on the number of guanosine-thymidine nucleotide repeats (GT) repeats that regulates expression levels and could have an impact on organ survival post-injury. We correlated HO-1 polymorphism with renal graft function. The HO-1 gene was sequenced (N = 181), and the allelic repeats were divided into subclasses: short repeats (S) (< 27 repeats) and long repeats (L) (>= 27 repeats). A total of 47.5% of the donors carried the S allele. The allograft function was statistically improved six months, two and three yr after transplantation in patients receiving kidneys from donors with an S allele. For the recipients carrying the S allele (50.3%), the allograft function was also better throughout the follow-up, but reached statistical significance only three yr after transplantation (p = 0.04). Considering only those patients who had chronic allograft nephropathy (CAN; 74 of 181), allograft function was also better in donors and in recipients carrying the S allele, two and three yr after transplantation (p = 0.03). Recipients of kidney transplantation from donors carrying the S allele presented better function even in the presence of CAN.

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPS) direct a myriad of developmental and adult homeostatic evens through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like. Kinasel and -2 (ALK1/ACVR1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp(+). In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sat mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by sax(P), a viable allele previously reported to be mill, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first: class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVR1-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.