Upstream Stimulating Factors 1 and 2 Enhance Transcription from the Placenta-Specific Promoter 1.1 of the Bovine Cyp19 Gene

<p>Abstract</p> <p>Background</p> <p>Placenta-derived oestrogens have an impact on the growth and differentiation of the trophoblast, and are involved in processes initiating and facilitating birth. The enzyme that converts androgens into oestrogens, aromatase cytochrome P450 (P450arom), is encoded by the <it>Cyp19 </it>gene. In the placenta of the cow, expression of <it>Cyp19 </it>relies on promoter 1.1 (P1.1). Our recent studies of P1.1 <it>in vitro </it>and in a human trophoblast cell line (Jeg3) revealed that interactions of placental nuclear protein(s) with the E-box element at position -340 are required for full promoter activity. The aim of this work was to identify and characterise the placental E-box (-340)-binding protein(s) (E-BP) as a step towards understanding how the expression of <it>Cyp19 </it>is regulated in the bovine placenta.</p> <p>Results</p> <p>The significance of the E-box was confirmed in cultured primary bovine trophoblasts. We enriched the E-BP from placental nuclear extracts using DNA-affinity Dynabeads and showed by Western blot analysis and supershift EMSA experiments that the E-BP is composed of the transcription factors upstream stimulating factor (USF) 1 and USF2. Depletion of the USFs by RNAi and expression of a dominant-negative USF mutant, were both associated with a significant decrease in P1.1-dependent reporter gene expression. Furthermore, scatter plot analysis of P1.1 activity <it>vs. </it>USF binding to the E-box revealed a strong positive correlation between the two parameters.</p> <p>Conclusion</p> <p>From these results we conclude that USF1 and USF2 are activators of the bovine placenta-specific promoter P1.1 and thus act in the opposite mode as in the case of the non-orthologous human placenta-specific promoter.</p

On the relevance of technical variation due to building pools in microarray experiments

Background Pooled samples are frequently used in experiments measuring gene expression. In this method, RNA from different individuals sharing the same experimental conditions and explanatory variables is blended and their concentrations are jointly measured. As a matter of principle, individuals are represented in equal shares in each pool. However, some degree of disproportionality may arise from the limits of technical precision. As a consequence a special kind of technical error occurs, which can be modelled by a respective variance component. Previously published theory - allowing for variable pool sizes - has been applied to four microarray gene expression data sets from different species in order to assess the practical relevance of this type of technical error in terms of significance and size of this variance component. Results The number of transcripts with a significant variance component due to imperfect blending was found to be 4329 (23 %) in mouse data and 7093 (49 %) in honey bees, but only 6 in rats and none whatsoever in human data. These results correspond to a false discovery rate of 5 % in each data set. The number of transcripts found to be differentially expressed between treatments was always higher when the blending error variance was neglected. Simulations clearly indicated overly-optimistic (anti-conservative) test results in terms of false discovery rates whenever this source of variability was not represented in the model. Conclusions Imperfect equality of shares when blending RNA from different individuals into joint pools of variable size is a source of technical variation with relevance for experimental design, practice at the laboratory bench and data analysis. Its potentially adverse effects, incorrect identification of differentially expressed transcripts and overly-optimistic significance tests, can be fully avoided, however, by the sound application of recently established theory and models for data analysis

Elevated free fatty acids affect bovine granulosa cell function: a molecular cue for compromised reproduction during negative energy balance

High-yielding dairy cows postpartum face the challenge of negative energy balance leading to elevated free fatty acids levels in the serum and follicular fluid thus affecting the ovarian function. Here, we investigated effects of physiological concentrations of palmitic acid (PA), stearic acid (SA) and oleic acid (OA) on the viability, steroid production and gene expression in a bovine granulosa cell (GC) culture model. Treatment with individual and combined fatty acids increased the CD36 gene expression, while no significant apoptotic effects were observed. Both PA and SA significantly upregulated the expression of FSHR, LHCGR, CYP19A1, HSD3B1, CCND2 and increased 17β-estradiol (E2) production, while OA downregulated the expression of these genes and reduced E2. Interestingly, STAR was equally downregulated by all fatty acids and combination treatment. E2 was significantly reduced after combination treatment. To validate the effects of OA, in vivo growing dominant follicles (10–19 mm) were injected with bovine serum albumin (BSA) with/without conjugated OA. The follicular fluid was recovered 48 h post injection. As in our in vitro model, OA significantly reduced intrafollicular E2 concentrations. In addition, expression of CD36 was significantly up- and that of CYP19A1 and STAR significantly downregulated in antral GC recovered from aspirated follicles. The ovulation rates of OA-injected follicles tended to be reduced. Our results indicate that elevated free fatty acid concentrations specifically target functional key genes in GC both in vitro and in vivo. Suggestively, this could be a possible mechanism through which elevated free fatty acids affect folliculogenesis in dairy cows postpartum

Phosphorylation of a Central Clock Transcription Factor Is Required for Thermal but Not Photic Entrainment

Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk(out) flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression

Temporal changes in clinical and radiographic variables in dogs with preclinical myxomatous mitral valve disease: The EPIC study

The Evaluation of pimobendan in dogs with cardiomegaly caused by preclinical myxomatous mitral valve disease (EPIC) study monitored dogs with myxomatous mitral valve disease (MMVD) as they developed congestive heart failure (CHF)

Distribution Analysis of Hydrogenases in Surface Waters of Marine and Freshwater Environments

Background Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism in marine environments is nitrogen fixation. Principal Findings We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H2 uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean. Significance This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H2-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives