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Differentiation-specific action of orphan nuclear receptor NR5A1 (SF-1): transcriptional regulation in luteinizing bovine theca cells
Norbert Walther1,2,
Martina Jansen1,3,
Wasima Akbary1,4 &
Richard Ivell1,5
The orphan nuclear receptor NR5A1 (steroidogenic factor-1, SF-1) is a master regulator of tissue-specific gene expression in reproductive and steroidogenic tissues. Two activating functions, AF-1 and AF-2, have been described to function in a cooperative manner to recruit transcriptional coactivators to the promoter regions of NR5A1-controlled genes.
The role of the NR5A1 activating functions AF-1 and AF-2 was studied in primary bovine theca cells. Bovine theca cells were infected with recombinant adenovirus vectors over-expressing wild-type NR5A1 or NR5A1 mutants, in which one of the activating functions of this orphan nuclear receptor had been impaired. Under different culture conditions, theca cell-specific transcript levels were measured by reverse transcription and real-time PCR.
Under culture conditions optimized for cell growth, transcriptional up-regulation of CYP11A1 (P450 side chain-cleavage enzyme) and INSL3 (Insulin-like factor 3, Relaxin-like factor (RLF)) was found to be dependent on the presence of NR5A1 carrying an intact AF-2. Under conditions inducing luteal differentiation of theca cells, CYP11A1 and STAR (Steroidogenic acute regulatory protein) were up-regulated by the action of luteinizing hormone (LH), whereas the differentiation-specific up-regulation of INSL3 was suppressed by LH in luteinizing theca cells. Inhibition of insulin- or IGF1- (insulin-like growth factor I) dependent signal transduction by the RAF1 kinase inhibitor GW5074 and the mitogen-activated protein kinase kinase inhibitor PD98059 resulted in the finding that RAF1 kinase inhibition was able to counteract the LH-dependent regulation of NR5A1-controlled genes, whereas inhibition of the mitogen-activated protein kinase (MAP kinase) pathway did not have any significant effect.
The regulation of the three NR5A1-controlled genes CYPA11, STAR, and INSL3 in luteinizing theca cells apparently is not dependent on NR5A1 activating functions AF-1 or AF-2. Activation of AF-1 here even appears to have an impairing effect on NR5A1 transcriptional activity, implying that up-regulation of NR5A1-controlled genes uses a different pathway. Our results might be explained by the possible existence of an interconnection between the RAF1 kinase and the cyclic AMP-protein kinase A pathway. Such a non-classical regulatory pathway might play an important role in the control of gene expression in reproductive and steroidogenic tissues.
The various differentiated phenotypes of cells and tissues are based upon the establishment of stable cell type-specific patterns of gene expression. The recent advances in the sequencing of mammalian genomes [1], together with the development of efficient high-throughput technologies for quantification of gene products [2, 3], allows the assessment of the tissue-specific expression levels for virtually all genes in a single experiment. Although data on gene expression can now be generated with high efficiency, sophisticated molecular biology techniques like chromatin immunoprecipitation [4] have brought to light completely new levels of complexity in the mechanisms controlling transcription [5, 6]. The elucidation of the regulatory functions of nuclear receptors represents a good example of the step-by-step detection of further levels of complexity. The nuclear receptor proteins purified first could be described as transcriptional activators controlled by small lipophilic molecules as ligands [7–9]. However, related proteins could be characterized, whose transcriptional activity apparently is not primarily controlled by ligand binding [10, 11]. These orphan nuclear receptors are evolutionary old molecules with essential roles in the regulation of development and tissue function. The principal effect of binding of these proteins to the promoters of controlled genes appears to be the generation of specific docking sites for transcriptional coregulators. The transcriptional activity of orphan nuclear receptors can be regulated by phosphorylation [12, 13]. Although recently in a number of cases small lipophilic molecules have been shown to be able to bind as ligands to orphan nuclear receptors [14–17], the functions of these compounds in vivo have not been well established. An additional level of transcriptional regulation by orphan nuclear receptors is the alternative binding to the same promoter sites [18, 19]. Binding of different orphan nuclear receptors can lead to the recruitment of different coactivators or corepressors to the promoter of the controlled genes [20, 21]. The subsequent process of gene activation comprises the establishment of an open chromatin conformation and the assembly of the RNA polymerase II transcriptional initiation complex [22–24]. The activities of the coactivators in this process, like the transcriptional activities of the orphan nuclear receptors, can be under the control of protein phosphorylation, modulating the specific interactions of coactivators with different transcriptional initiation complexes [25].
For the orphan nuclear receptor NR5A1, the carboxyterminal activating function AF-2, corresponding to the ligand-dependent activating function of nuclear hormone receptors, has been shown to be responsible for binding of co-activators of the p160 class providing an essential function for the constitutive transcriptional activity of NR5A1 [20]. The unique activating function AF-1 of NR5A1 located in the hinge region of the protein is regulated by mitogen-activated kinase (MAPK) phosphorylation and is involved in interactions with various cofactors and in stabilization of the AF-2 activating function [12, 26, 27]. In the light of the complexity of the mechanisms regulating transcription, the multifaceted effects of hormones and growth factors on the development and function of tissues are not surprising. For example, ovarian follicular cells are under cyclic control by the differentiation-inducing gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH), in combination with essential growth-promoting effects by hormones and growth factors like insulin and/or IGF1 (insulin-like growth factor I) [28, 29]. Tissue culture of bovine theca cells provides an excellent model system to investigate the influences of these endocrine and paracrine stimuli on the development of the differentiated phenotype or the induction of theca cell differentiation-specific genes [30]. Synergistic activation by LH and IGF1 appears to provide the full stimulus for luteal differentiation of ovarian follicular cells [31, 32]. Many of the genes induced or up-regulated during the luteinization process, for example the genes coding for the steroidogenic P450 enzymes, the Steroid Acute Regulatory Protein (STAR) and the Insulin-like factor 3 (INSL3) appear to be controlled by binding of the tissue-specific orphan nuclear receptor NR5A1 to specific binding sites in the promoter regions [33–39]. In the study presented here, the regulatory mechanisms controlling expression of these luteinization-specific genes were investigated, using in vitro cultures of bovine theca cells under conditions optimized for growth or differentiation.
Our results suggest that the established activating functions of NR5A1 only play a minor role in the control of reproductive functions during theca cell luteinization. Possibly, in luteinizing bovine theca cells an interconnection exists between the MAP kinase pathway and the cyclic AMP-protein kinase A pathway, constituting a non-classical pathway of growth factor and hormone action.
Plasmids, bacteria, cell lines, and chemicals
Expression vectors for full-length wild-type NR5A1 cDNAs (pRSV/Ad4BP (bovine NR5A1) and pCMV119+ (murine NR5A1)) were kind gifts of Dr. K.I. Morohashi (Okazaki, Japan) and Dr. K. Parker (Dallas, TX), respectively. The bovine AF-2 deletion mutant (pAd4BP/ΔAF-2) was kindly provided by Dr. J. Lund (Bergen, Norway), the murine AF-1 mutant S203A (pCI-HA-NR5A1) was generously supplied by Dr. H.A. Ingraham (San Francisco, CA). The components of the AdEasy adenoviral expression system, plasmids pAdEasy-1 and pAdTrack-CMV, as well as the rec+ bacterial strain E. coli BJ5183, were obtained from Dr. T. Wieland (Mannheim, Germany) with the kind permission of Dr. B. Vogelstein (Baltimore, MD). HEK 293 cells for adenovirus propagation were a gift of Dr. F. Schnieders (Hamburg, Germany), HeLa cells were obtained from the American Type Culture Collection. Both cell lines were cultivated in Dulbecco minimum essential medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and fetal calf serum (10 % for HeLa cells, varying amounts ranging from 2 % to 5 % for HEK 293 cells). Protein kinase inhibitors H89 and PD98059 were purchased from Calbiochem (San Diego, CA), inhibitor GW5074 and all other chemicals were from Sigma (Munich, Germany).
Construction of NR5A1-expressing recombinant adenoviruses
All recombinant DNA experiments were performed using standard laboratory protocols [40]. Wild-type bovine NR5A1 cDNA was directly recloned into shuttle plasmid pAdTrackCMV [41] as a HindIII/XbaI fragment. The cDNA coding for the AF-2 deletion mutant was released by digestion and blunting at the BamHI site, followed by SalI cleavage; the resulting fragment was inserted into SalI/EcoRV-digested pAdTrackCMV. Kanamycin-resistant plasmid clones were analysed by NdeI cleavage. Wild-type murine NR5A1 cDNA was isolated as an EcoRI fragment and, after subcloning into pBSSK (Stratagene, Heidelberg, Germany), released by digestion with KpnI and XbaI and inserted into appropriately cleaved pAdTrackCMV. The cDNA coding for the S203A mutant was directly recloned as XhoI/NotI fragment into SalI/NotI-digested pAdTrackCMV. Identity and orientation of NR5A1 sequences was confirmed by dideoxy sequencing. As an empty virus vector control pAdTrackCMV was used without any further modification.
For generation of complete adenoviruses by homologous recombination, the NR5A1 wild-type and mutant shuttle plasmids were linearized by PmeI digestion and purified by agarose gel electrophoresis to minimize the contamination with circular plasmid DNA. 80 to 1200 ng of these DNAs, together with 100 ng of undigested plasmid pAdEasy-1 comprising the necessary additional adenoviral sequences [41], were transfected into rec+ E. coli BJ5183 by electroporation. Cuvettes with 2 mm distance between electrodes were used with the Gene Pulser System (BIORAD, Munich, Germany) at settings of 2500 V, 25 μF, and 200 Ω, resulting in time constants of the pulse of approximately 5 msec. Directly after electroporation, bacteria were incubated at 37°C for 1 hour with shaking and spread on kanamycin-containing plates. Recombinant adenoviral plasmid clones were selected after 16 hours at 37°C as small colonies. Plasmid DNA from these clones was analysed by digestion with PacI, plasmids containing the expected 3 kb PacI fragment were retransformed into rec- E. coli for further analysis and preparation of adenoviral DNA. The identity of recombinant adenovirus constructs was confirmed by diagnostic PCR using different combinations of primers derived from adenovirus vector or NR5A1 sequences and by dideoxy sequencing.
Preparation of infectious adenovirus particles
Recombinant adenoviral plasmid DNAs were linearized by PacI digestion leading to the excision of plasmid sequences and used for adenovirus propagation. Four to 25 μg of adenovirus DNAs were transfected into HEK 293 cells grown in T25 tissue culture flasks to approximately 70 % confluence by lipofection using the Polyfect reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Virus expression was monitored by fluorescence microscopy of the coexpressed green fluorescent protein. Seven to 10 days after transfection, cells were scraped off the flask and cells and supernatants were subjected to three cycles of freezing and thawing for release of virus particles from infected cells. Cellular debris was removed by centrifugation (5 min, 3800 rpm) and total supernatants were used for infection of T75 flasks of HEK 293 cells grown to 70 % confluence. After 2 days virus suspensions were prepared from the infected cells and total supernatants used for infection of T175 flasks of HEK 293 cells as described above. The titer of the virus suspensions prepared from these cells was determined by limiting dilution using HEK 293 cells. For the final step of adenovirus propagation, 5 T300 tissue culture flasks were seeded each with 2 × 107 HEK 293 cells and the cells infected with recombinant adenoviruses at a multiplicity of infection of 25 (5 × 108 plaque-forming units per flask). Approximately 2 days after infection, when infected cells just began to detach from the plate, supernatants were carefully removed. The cells were scraped off the flask and subjected to freeze/thaw lysis as described above. After centrifugation, 5 ml each of the virus suspensions were layered on top of CsCl step gradients (2 ml 1.45 g/cm3, 2 ml 1.35 g/cm3, 1 ml 1.25 g/cm3) in Beckman SW40 ultracentrifuge tubes. Tubes were filled up and tared with mineral oil and centrifuged for 2 hours at 35,000 rpm and 10°C. After removal of cellular and viral debris, intact virus particles were collected using a 1 ml syringe and transferred to new SW40 tubes. Tubes were filled up with CsCl solution (1.35 g/cm3), tared with mineral oil, and centrifuged for 20 hours at 30,000 rpm and 4°C. The virus band was collected as described above and desalted by passage through a NAP-25 gel filtration column (Amersham Pharmacia Biotech, Frankfurt, Germany) using virus storage buffer (10 mM Tris/HCl, pH 8.0, 135 mM NaCl, 3 mM KCl, 1 mM MgCl2, 10 % glycerol). The virus suspensions were divided into aliquots and stored at -80°C. The titer of the frozen stocks was determined by limiting dilution using HEK 293 cells.
Preparation and culture of bovine theca cells
For preparation of bovine theca cells, the established protocol described previously by Brunswig-Spickenheier and Mukhopadhyay [30] was used. Briefly, bovine ovaries were collected at a local slaughterhouse and healthy tertiary follicles with a diameter of 10 to 25 mm were used for dissection of the theca interna layer. After enzymatic dissociation of the tissue, theca cells were purified by centrifugation over Percoll (density 1.07 g/cm3). Theca cells were suspended in a 1:1 mixture of Dulbecco minimum essential medium and Ham F12 medium containing 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (basal medium) and 5 × 105 cells per well seeded into 6-well tissue culture plates (Nunc, Wiesbaden, Germany) coated with bovine dermal collagen (Cellon, Strassen, Luxembourg). For the investigation of growing cultures, the basal medium was supplemented with 2.5 % fetal calf serum. After 2 days of culture at 37°C under an atmosphere of 95 % air and 5 % CO2, the medium was changed to basal medium supplemented with 10 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, and 0.1 % bovine serum albumin. For long-term cultures of differentiating theca cells, the basal medium was supplemented with 100 ng/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, and 0.1 % bovine serum with or without the addition of 10 ng/ml bovine luteinizing hormone (bLH, kind gift of the NIADDK and the National Hormone and Pituitary Program, NIH, Bethesda, MD); the medium was changed in regular intervals every 2 days.
Infection of bovine theca cells with NR5A1 expressing recombinant adenoviruses
Prior to adenovirus infection, medium was changed on theca cell cultures adding only 1 ml of basal medium per well. Diluted virus stocks were then added with a multiplicity of infection of 100 (5 × 107 plaque-forming units per well) and evenly distributed by gentle shaking immediately after addition. After incubation for 30 min, 1.5 ml of basal medium and the necessary supplements were added and the infected cells cultured for another 2 or 3 days. Adenovirus infections of growing theca cells were performed at day 5 of culture, infections of long-term cultures were performed at days 5, 8, and 11. Virus expression was monitored by fluorescence microscopy of the coexpressed green fluorescent protein. For isolation of total cellular RNA, cells were lysed 48 to 72 hours after infection in 500 μl peqGOLD RNAPure reagent (Peqlab, Erlangen, Germany) per well and RNA purified following the manufacturer's instructions and stored at -80°C. For isolation of total cellular protein, cells were lysed in 250 μl RIPA buffer (10 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1 % SDS, 1.0 % Triton X-100, 1.0 % Na-deoxycholate, 1 × Complete Protease Inhibitor Cocktail (Roche Diagnostics, Penzberg, Germany)). After removal of cellular debris by centrifugation, protein extracts were aliquoted and stored at -80°C.
Quantification of theca cell-specific transcripts
1 to 2 μg total bovine theca cell RNA were reverse transcribed using SuperScript II RNase H- reverse transcriptase (Invitrogen, Karlsruhe, Germany) and random hexamer oligonucleotide primers (Amersham Biosciences, Freiburg, Germany) according to the manufacturer's instructions. cDNAs were diluted 1 : 5 with ultra-pure water and stored at -20°C. For amplification of theca cell-specific transcripts, real time-PCR measurements were performed in triplicate on the LightCycler System (Roche Diagnostics). PCR reactions were set up using the QuantiTect SYBR Green PCR Kit (Qiagen) and specific oligonucleotide 5' and 3' primers (10 pmol each). Primer sequences for amplification of partial cDNA sequences of theca cell-specific genes were selected using the Primer3 PCR primer picking program [42]. The primer sequences used are listed in Tab. 1. For initial denaturation and activation of the DNA polymerase, samples were heated to 95°C for 15 min. Amplifications were performed over up to 40 cycles (95°C 15 sec (denaturing), 50°C 30 sec (annealing), 72°C 15 sec (polymerisation), 75°C 5 sec (data acquisition)). The specificity of the reactions was monitored by melting curves of the products and agarose gel electrophoresis, and the identity of the products confirmed by dideoxy sequencing. Standard curves for measurements of PCR efficiencies were generated using serial 1 : 5 dilutions of pooled theca cell cDNAs. Expression of the ribosomal protein RPS27A was used as an internal control for relative transcript quantification. For the calculation the method of Pfaffl [43] was used, including an automatic correction for differences in PCR efficiencies. Testing for statistical significance of the differences in transcript levels was performed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test using the GraphPad Prism 4.0 software package (GraphPad Software Inc., San Diego, CA); P < 0.05 was considered statistically significant.
Table 1 Primer sequences used for amplification of bovine theca cell transcripts
GFP fluorescence of adenovirus-infected theca cells was visualised by fluorescence microscopy using a Diaphot microscope (Nikon, Düsseldorf, Germany) equipped with a DM510 filter block and a 10DIC lens at 100-fold magnification. Digital images were recorded using a Leica DC200 camera (Leica Microsystems, Wetzlar, Germany) and stored as JPEG files for documentation.
Northern blot and Western blot analyses
Total cellular RNA or protein was isolated 48 hours after infection. In vitro translation of NR5A1 proteins was performed using the TnT rabbit reticulolysate system (Promega, Mannheim, Germany). Northern blot analysis of adenovirus-infected HeLa cells was performed as described previously [44]. DNA fragments comprising bovine NR5A1 or human ribosomal protein RPS27A coding sequences were radiolabelled and used as probes. For Western blot analysis 6 μg of total cellular proteins were separated on a 10 % NuPAGE gel (Invitrogen, Karlsruhe, Germany) at 200 V and blotted to a PVDF membrane at 30 V following the manufacturer's instructions. After blocking with 5 % non-fat milk powder, as primary antibody a rabbit antiserum directed against the bovine NR5A1 protein (kind gift of K.I. Morohashi, dilution 1 : 5000) was allowed to bind over-night at 4°C. After washing, the second antibody (horseradish-peroxidase-conjugated anti-rabbit IgG (Dianova, Hamburg, Germany) was added at a dilution of 1 : 5000 and allowed to bind for 3 hours at room temperature. After final washing, binding of antibodies was analyzed using the Amersham ECL Plus detection system (GE Healthcare, Munich, Germany).
Adenoviral vectors for over-expression of NR5A1 wild-type and mutants
The orphan nuclear receptor NR5A1 is one of the most important regulators of tissue-specific gene expression in reproductive and steroidogenic tissues. Although the binding of this tissue-specific transcription factor to regulatory sites in the promoters of numerous genes expressed in these tissues has been established [39], the mechanisms by which NR5A1 exerts control of transcription remain largely unelucidated. In the study presented here, an experimental system has been established that allows the analysis of NR5A1 action during growth and differentiation of gonadal cells. Bovine theca cells were cultivated under different in vitro conditions and infected with recombinant adenovirus vectors over-expressing wild-type NR5A1 or NR5A1 mutants, in which one of the activating functions of this orphan nuclear receptor had been impaired. This approach allowed us to measure separately the effects mediated by the individual NR5A1 activating functions on the transcription of tissue-specifically expressed genes in theca cells during growth and differentiation.
NR5A1 has been shown to contain two activating functions. A mutant carrying a deletion of the core region of the AF-2 activating function has been shown to suppress completely the activation of steroidogenic enzyme expression [45]. The recently defined unique AF-1 activating function of NR5A1 has been shown to be regulated by phosphorylation of amino acid serine 203 [12] leading to a stabilization of the active conformation of NR5A1 [27]. Complete cDNAs for wild-type bovine as well as murine NR5A1, a mutant carrying the described deletion of the AF-2 core region (ΔAF-2), and a mutant carrying a substitution of the phosphorylatable amino acid serine 203 by alanine (S203A) (Fig. 1A) were used for generation of recombinant adenoviruses over-expressing these different NR5A1 proteins in infected cells.
Transactivation domains of NR5A1 wildtype and mutants over-expressed in bovine theca cells. In the ΔAF-2 mutant the essential core region of the AF-2 activating function has been deleted. In the AF-1 mutant amino acid serine 203 has been substituted by alanine preventing the MAP kinase-dependent phosphorylation of this site (A). Northern blot analysis of wild-type and mutant mRNAs transcribed from NR5A1-expressing recombinant adenoviruses in infected HeLa cells (Control: AdEasy adenoviral vector without insert). As a control for equal RNA loading a probe specific for human ribosomal protein RPS27A was used (B). Western blot analysis of NR5A1 protein from adenovirus-infected HeLa cells and rabbit reticulocyte lysate in vitro translation using an antiserum directed against bovine NR5A1 (Control: AdEasy adenoviral vector without insert) (C).
In the AdEasy adenovirus vector system [41] used in this study the inserted genes of interest are expressed to high levels of mRNA from the strong ubiquitously active cytomegalovirus immediate early promoter. Infections of HeLa cervical carcinoma cells were performed to obtain mRNA amounts large enough to perform Northern blot analysis to check for size and stability of the transcripts (Fig. 1B). NR5A1 cDNA sequences are expressed to high levels of stable mRNAs whose sizes correspond well with the sizes of the open reading frames contained within the cloned inserts. Adenovirus vectors expressing NR5A1 wild-type and mutant mRNAs were further tested for NR5A1 protein expression. In parallel, Western blot analyses of infected HeLa cervical carcinoma cells using an antiserum specific for bovine NR5A1 [46] were performed, demonstrating the expression of a major protein band with an estimated molecular weight of approximately 52 kDa comigrating with bovine NR5A1 protein in vitro translated from the original plasmid expression vector (Fig. 1C).
Over-expression of NR5A1 wild-type and mutants in growing bovine theca cells
The main stimulus inducing luteal differentiation of theca cells is the secretion of luteinizing hormone (LH) to high levels around the time of ovulation. The acute cellular response of theca cells to this stimulus is the increase in the intracellular concentration of the second messenger cyclic AMP, leading within hours to a total reorganization of the actin cytoskeleton of the cell generally referred to as "stellation", which is characterized by cytoplasmic retraction and the formation of narrow, elongated cellular processes [47]. This response was investigated in bovine theca cells infected with recombinant adenoviruses over-expressing wild-type or mutant NR5A1 (Fig. 2). Infected cells were stimulated or not with a combination of insulin and the adenylate cyclase activator forskolin and visualised by the fluorescence of the green fluorescent protein coexpressed from the recombinant adenoviruses. Although the morphology of the theca cells undergoing "stellation" is variable, nearly all cells in the cultures show this response, the cytoplasmic retraction being indicated by the increased level of perinuclear fluorescence. The results show that previous infection and over-expression of wild-type or mutant NR5A1 did not influence the acute cellular response of bovine theca cells to the luteinizing stimulus.
GFP fluorescence of bovine theca cells infected with adenoviral vectors expressing NR5A1. Bovine theca cells were infected with adenoviral vectors expressing wild-type or mutant NR5A1 and stimulated or not (control) with 2 μg/ml insulin and 10 μM forskolin (Insulin/Forskolin). The scale bar represents 20 μm.
The results of the initial experiments demonstrate that infection with recombinant adenoviruses at a multiplicity of infection of 100 induces expression of wild-type and mutant NR5A1 to high levels in bovine theca cells without toxic effects and without disturbing intracellular responses. The high multiplicity is needed in order to obtain infection of 100 % of the cells in the culture, probably due to variations in the number of virus receptors on the surfaces of individual theca cells. Adenovirus infections of bovine theca cells were performed to elucidate the contribition of the two NR5A1 activating functions to the transcriptional regulation of theca cell-specifically expressed genes. Growing cultures of bovine theca cells were infected at day 5 of culture, total RNA isolated 2 days after infection, and relative transcript levels quantificated by real time-PCR (Fig. 3). Both the bovine and the murine NR5A1 wild-type forms were used because of the different genetic backgrounds of the two NR5A1 mutants investigated. The results show for the steroidogenic enzyme gene CYP11A1, a classical NR5A1-regulated gene [35, 48], a massive up-regulation of transcription by over-expression of wild-type NR5A1, indicating that this gene is not fully activated by the endogenous NR5A1. This transcriptional activation is completely abolished in the cultures over-expressing the NR5A1 mutant lacking the AF-2 activating function (ΔAF-2), known to be important for coactivator binding. Western blot analysis of NR5A1 proteins confirmed that the mutant protein was expressed to levels identical to those of wild-type NR5A1 (see Fig. 1C). On the contrary, mutation of the MAP kinase phosphorylation site (S203A), known to be essential for AF-1 activating function, has no effect whatsoever on the transcriptional activation of CYP11A1 induced by NR5A1 over-expression. This finding suggests that in bovine theca cells, in contrast to other experimental systems [12], MAP kinase phosphorylation of the NR5A1 AF-1 activating function does not effect NR5A1 function. The observed pattern of AF-2 dependent activation is not restricted to the steroidogenic enzyme gene CYP11A1. The transcriptional activation of the tissue-specific growth factor INSL3 (Insulin-like factor 3) by over-expressed NR5A1 follows the same pattern, albeit at lower levels. This NR5A1-controlled promoter [39] apparently is regulated by the same principal mechanism that is governing CYP11A1 expression. Transcription from the promoter of the STAR (Steroidogenic acute regulatory protein) gene does not follow this pattern of activation by NR5A1; this gene does not appear to be affected by NR5A1 over-expression. The differences between the STAR expression in the cultures over-expressing bovine or murine wild-type NR5A1, although statistically significant, have been found to be not reproducible in parallel experiments. The promoter of the bovine STAR gene can be up-regulated by NR5A1 over-expression in transfection experiments [37]. Several sequences in the bovine STAR promoter show reasonably good matches with the established NR5A1 binding site, but binding of NR5A1 to the bovine STAR promoter was found to be relatively weak in this study. In the light of these findings, the results presented here indicate that transcription of the STAR gene, despite its expression in the same tissues as CYP11A1, may not be regulated by direct binding of NR5A1 to the promoter and subsequent coactivator recruitment, but by a different, yet uncharacterized, mechanism.
Transcriptional regulation by NR5A1 of theca cell marker genes in growing cultures. Bovine theca cells cultivated in the presence of 10 μg/ml insulin for maximal growth were infected with adenoviral vectors expressing bovine or murine wild-type NR5A1 (WT) or mutants lacking one of the two activating functions (ΔAF-2 or S203A, control: AdEasy adenoviral vector without insert). CYP11A1, STAR, and INSL3 transcripts were quantified by real time-PCR as described in Materials and Methods. The results of one representative out of three experiments are shown. Data are expressed as mean ± standard deviation. The groups designated by the letters above the bars differ significantly at the level of P < 0.05.
Over-expression of NR5A1 wild-type and mutants in long-term cultures of differentiating bovine theca cells
In order to investigate the transcriptional regulation of theca cell-specifically expressed genes under conditions that resemble better their up-regulation in vivo, long-term cultures of bovine theca cells were set up in the presence or the absence of LH as the natural luteinizing stimulus (Fig. 4). Under these established conditions, the time-course of luteal differentiation of theca cells can be observed in tissue culture [44]. The results show that, in spite of the different regulatory effects of NR5A1 on the CYP11A1 and the STAR promoter, expression of both genes is totally dependent on stimulation by LH and is increasing during prolonged culture, following essentially identical time courses. On the contrary, expression of INSL3 slowly decreases during prolonged culture under the influence of LH. In cultures without addition of LH, however, INSL3 expression starts to rise after more than 8 days, with levels still increasing after 14 days of culture, confirming the results of a previous study [44]. Apparently in this case, in spite of the same mechanism of NR5A1 regulation of the INSL3 and CYP11A1 promoters in growing cells, the expression of INSL3 during theca cell luteinization is controlled by a principally different mechanism.
Time-course of marker gene expression during differentiation of bovine theca cells in culture. Long-term cultures of bovine theca cells containing minimal amounts of insulin (100 ng/ml) were set up with (filled circles) or without (open circles) the addition of 10 ng/ml bovine luteinizing hormone (LH). At the time-points indicated, CYP11A1, STAR, and INSL3 transcripts were quantified by real time-PCR as described in Materials and Methods. The results of one representative out of three experiments are shown. Data are expressed as mean ± standard deviation. Significant differences (P < 0.05) to the unstimulated cultures at day 8 are indicated by asterisks.
Infections of long-term cultures of differentiating bovine theca cells (days 5, 8, and 11 of culture) were performed in order to analyze the effects of NR5A1 wild-type and mutant over-expression. At day 11, expression of CYPA11 and STAR was induced by LH 8-fold and 16-fold, respectively, whereas the induction of INSL3 was totally abolished by addition of the hormone, leading to a 6-fold reduction of the INSL3 expression level (see Fig. 4). Results are presented for the infections at day 11 (Fig. 5), the infections at day 5 and 8 yielded essentially the same results. The presentation of the normalized results of the infection experiments levels out the stimulation or inhibition of expression by LH, displaying solely the transcriptional effects of NR5A1 over-expression. In the cultures without addition of LH, robust elevation of transcript levels by NR5A1 over-expression could be observed for CYP11A1 demonstrating the function of the adenovirally expressed NR5A1. Interestingly, this increase was found in all cultures, irrespective of the status of the activating functions of the over-expressed NR5A1 proteins. Apparently the established activating functions AF-1 and AF-2 of NR5A1 both are not essential for the exertion of its constitutive transcriptional activation under these conditions. The variations between the cultures in the levels of STAR and INSL3 expression, although statistically significant, have been found to be not reproducible in parallel experiments, suggesting no specific effect of NR5A1 on expression of these genes in the absence of LH. The relative expression levels of theca cell-specific transcripts in the cultures with addition of LH and infected with adenoviruses expressing bovine or murine wild-type NR5A1 or the mutant lacking the AF-2 activating function were variable and, in the case of CYP11A1 transcripts, not reproducible in parallel experiments. The elevation of STAR and INSL3 expression levels in these cultures was maximally two-fold compared to LH-stimulated control cultures. However, infection of theca cells with the AF-1 activating function mutant unable to be phosphorylated by MAP kinases consistently led to a higher induction of CYP11A1, STAR, and INSL3 transcription. This suggests that phosphorylation at this site might have a detrimental effect on the potency of NR5A1 as a transcriptional activator in certain promoter or cellular contexts. This effect is most clearly demonstrated at the INSL3 promoter, which in the presence of LH is down-regulated. Presumably, in the differentiating theca cells, NR5A1 phosphorylation at serine 203 leads to the inactivation of a yet uncharacterized activating function of this protein. Our finding differs substantially from the effects of the NR5A1 activating function AF-1 described in other experimental systems. This effect of serine 203 phosphorylation will have to be investigated in further studies.
Transcriptional regulation by NR5A1 of theca cell marker genes during differentiation in culture. Bovine theca cells were cultivated for 11 days in the presence of minimal amounts of insulin (100 ng/ml) with or without the addition of 10 ng/ml bovine luteinizing hormone (LH) and subsequently infected with adenoviral vectors expressing bovine or murine wild-type NR5A1 or mutants lacking one of the two activating functions (ΔAF-2 or S203A, control: AdEasy adenoviral vector without insert). 72 hours after infection, CYP11A1, STAR, and INSL3 transcripts were quantified by real time-PCR as described in Materials and Methods. The transcript levels in the NR5A1-infected cells are presented in relation to the levels in control-infected cells under identical culture conditions (with or without addition of LH). Thus the bars represent the transcriptional effects of over-expression of NR5A1 wild-type or mutants independent of the stimulatory or inhibitory effects of LH. The results of one representative out of three experiments are shown. Data are expressed as mean ± standard deviation. The groups designated by the letters above the bars differ significantly at the level of P < 0.05.
Investigation of the role of protein kinase pathways in the regulation of NR5A1-controlled genes
For investigation of the role of the MAP kinase pathway in regulation of NR5A1 function, experiments were performed aimed at the dissection of the kinase cascade constituting this regulatory pathway in bovine theca cells. Long-term cultures of bovine theca cells after 11 days of culture in the presence of LH were treated with specific inhibitors for RAF1 kinase (GW5074) or MAP kinase-kinase (PD98059) as sequential steps of the MAP kinase pathway, or an inhibitor for the cyclic AMP-dependent protein kinase A (H89) (Fig. 6). No cytotoxic effects of the inhibitors were observed over the concentration range employed in these experiments. Addition of the protein kinase A inhibitor H89, as expected, led to a down-regulation of CYP11A1 and STAR expression, the inhibitor counter-acting the intracellular effects of the exposure of the cells to exogenous LH. The dramatic increase in INSL3 expression under these conditions suggests that INSL3 expression is actively suppressed by a protein kinase A-dependent mechanism under conditions of LH stimulation.
Involvement of protein kinase pathways in the regulation of marker genes in differentiating theca cells. Bovine theca cells were cultivated for 11 days in the presence of minimal amounts of insulin (100 ng/ml) and 10 ng/ml bovine luteinizing hormone and subsequently treated with different amounts of specific inhibitors for protein kinase A (H89, squares), RAF1 kinase (GW5074, triangles) or MAP kinase-kinase (PD 98059, circles). After 72 hours, CYP11A1, STAR, and INSL3 transcripts were quantified by real time-PCR as described in Materials and Methods. The results of one representative out of three experiments are shown. Data are expressed as mean ± standard deviation. Significant differences (P < 0.05) to the uninhibited control cultures are indicated by asterisks.
A consistently observed result of this experiment is the inhibitory effect exerted by the RAF1 kinase inhibitor GW5074 on the expression of both the CYP11A1 and the STAR gene, in sharp contrast to the lack of any major effects of the MAP kinase-kinase inhibitor PD98059. The lack of correlation between the inhibition of sequential steps of the MAP kinase signal transduction pathway suggests that this pathway may not exist as an isolated entity in theca cells. The effect exerted by RAF1 kinase inhibition strongly correlates with the inhibition effected by the protein kinase A inhibitor H89. Possibly, in differentiating bovine theca cells there exists an interconnection between the MAP kinase and the cyclic AMP-protein kinase A pathways. Such an interconnection would allow RAF1 kinase to stimulate protein kinase A by phosphorylation of some upstream signalling component. This pathway of signal transduction would not be totally unprecedented, as RAF1 kinase-mediated phosphorylation of several adenylyl cyclase isoforms has recently been described in HEK 293 cells [49]. Our results show that such an interconnection between the two signalling pathways may be of importance in regulating luteal differentiation of theca cells. Further studies using complementary methods will help to elucidate this non-classical regulatory mechanism.
The orphan nuclear receptor NR5A1 has been shown to be one of the master regulators controlling the development and function of reproductive and steroidogenic tissues [50]. NR5A1-regulated genes as a whole are controlled by numerous different regimes of hormonal or developmental control [51], producing the high diversity of cellular phenotypes in the reproductive system. For example, the chromatin structure of the promoter region and the presence or absence of tissue-specifically expressed coregulators, both of fundamental importance for the regulation of gene expression in differentiation processes [52, 53], are not taken into account in standard transient transfection studies. On the contrary, bovine theca cells in primary culture as a population of steroidogenic cells from a reproductive tissue have been shown to express differentiated features during long-term culture under serum-free conditions [30]. Therefore these cells have been used in the study presented here for the investigation of NR5A1 function in a model system resembling theca cell luteinization in vivo. The use of adenoviral vectors for NR5A1 over-expression in bovine theca cells enabled us to transfer NR5A1 mutants with only one of the two activating functions impaired, and to study the effect of AF-1 and AF-2 on the expression of tissue-specifically expressed genes that are regulated during theca cell luteinization.
Three NR5A1-controlled genes whose expression had been shown to be regulated specifically during luteinization of bovine theca cells, CYP11A1 [54], STAR [37], and INSL3 [44], were selected for quantification of transcript levels. The up-regulation of CYP11A1 and STAR could be shown to be dependent on the presence of LH during long-term culture, in concordance with previous findings. The lack of major effects of NR5A1 on STAR expression, in contrast to the results of experiments using acute LH stimulation of bovine theca cells [56], suggests that, at this promoter, only the direct transcriptional effects of LH may be mediated by NR5A1, whereas the differentiation-specific up-regulation investigated here uses a different regulatory mechanism. NR5A1-mediated, LH-dependent up-regulation of transcription has been shown previously for the gene coding for the neuropeptide hormone oxytocin in the other type of steroidogenic cell in the bovine ovary, the granulosa cell [57]. The oxytocin promoter could be shown by us to be occupied by NR5A1 under this condition of active gene expression [18, 58, 59], demonstrating the important role of NR5A1 in the LH-dependent up-regulation of transcription. In contrast to the LH-dependency of CYP11A1 and STAR expression, INSL3 up-regulation during long-term culture of bovine theca cells was found to be impaired under LH stimulation. This finding is in perfect correlation with the results of our preceding study [44], confirming that INSL3 expression during bovine theca cell differentiation appears to be regulated by a different mechanism than the one controlling the expression of this gene in the male gonad. In the testis, INSL3 expression is up-regulated cell type-specifically in the steroidogenic Leydig cells, but in contrast to the situation in luteinizing bovine theca cells, this up-regulation is stimulated by LH [60].
The LH-dependent up-regulation of INSL3 expression in mouse Leydig cells appears to be dependent on binding of NR4A1 (NGFI-B) to a specific binding site in the INSL3 promoter [61]. This orphan nuclear receptor is involved in the acute LH-dependent up-regulation of gene expression in granulosa cells around the time of ovulation, and its expression apparently persists in the cells of the corpus luteum [62]. However, the NR4A1 binding site in the INSL3 promoter appears not to be conserved in all species, being absent in the bovine (Walther and Ivell, unpublished results). Apparently, there exist species-specific differences in the details of the mechanisms regulating INSL3 gene expression. The investigation of the contributions of the two NR5A1 activating functions, AF-1 and AF-2, to the control of tissue-specific gene expression in theca cells yielded some unexpected results: Firstly, a comparison of the transcriptional effects of AF-2 between growing bovine theca cells and bovine theca cells luteinizing in long-term cultures demonstrated that, although AF-2 is necessary for NR5A1-dependent up-regulation of gene expression in growing cells, this activating function apparently is dispensible for the exertion of the stimulatory effects of NR5A1 on gene expression during bovine theca cell luteinization. The second result was the total lack of effect of serine 203 phosphorylation on NR5A1 transcriptional activity. This finding implies that the MAP kinase pathway does not contribute directly to the activation of NR5A1-controlled genes by targeting the AF-1 activating function. However, luteal differentiation of bovine theca as well as granulosa cells has been shown to be dependent on stimulation of tyrosine receptor kinases by insulin or IGF1 [28, 44, 63]. These stimuli in many types of cells result in activation of the MAP kinase signal transduction pathway and subsequent activation of transcription factors like activator protein-1(AP-1) [64]. A possible explanation of our findings is that this pathway may not be functional in luteinizing bovine theca cells and that the undoubted stimulatory effects of insulin or IGF1 on these cells are exerted by a different pathway. Corresponding results have been obtained in a study investigating STAR gene expression in mouse Leydig cells, where the majority of the stimulatory effects of IGF1 are exerted via the protein kinase C pathway [65].
The results of our experiments suggest that, although the orphan nuclear receptor NR5A1 is necessary for the expression of tissue-specifically expressed genes that control the reproductive functions of the ovary, the established activating functions of NR5A1 play only a minor role, if any, in the control of gene expression during theca cell luteinization. Apparently, in luteinizing theca cells a different mechanism is involved in the regulation of NR5A1-controlled genes. The results of our experiments using specific inhibitors for the MAP kinase and the cyclic AMP-protein kinase A pathway might be explained by the possible existence of an interconnection between these two signal transduction pathways. The recent finding that RAF1 kinase can mediate the phosphorylation of several adenylyl cyclase isoforms [49] demonstrates that such interconnections exist and may be functional depending on cell type or differentiation state. The regulatory role of NR5A1 under these conditions appears to be somewhat reduced to the provision of coactivator docking sites. Transcriptional regulation of NR5A1-controlled genes in luteinizing theca cells presumably is exerted by specific modifications in the composition of coactivator complexes or modifications of the coactivators themselves. The main aim of this study using adenoviral expression of NR5A1 wild-type and mutants was to elucidate the contribution of the NR5A1 activating functions AF-1 and AF-2 to the transcriptional regulation of luteinization-specific genes. For subsequent studies, knock-down of the expression of the involved genes by short interfering RNA (siRNA) will be helpful in analyzing the details of the differentiation-specific mechanisms governing the expression of NR5A1-controlled genes.
In addition to the regulatory mechanisms discussed above, future studies on the regulation of orphan nuclear receptor-controlled genes will have to take into account the highly dynamic formation and destruction of promoter-nuclear receptor-coactivator complexes [5, 6]. This new level of complexity demands the development of new experimental methods in order to advance the classical models of transcriptional regulation. Evidence is accumulating that the intracellular signal transduction pathways do not exist in the cell as isolated entities, but are interconnected to an intricate regulatory network. These interconnections previously referred to as "crosstalk" apparently are of outmost importance as essential constituents of this network. RAF1 kinase presumably assumes a central role in interconnecting the "growth promoting" MAP kinase pathway to the "differentiation promoting" cyclic AMP-protein kinase A pathway [66], thereby contributing to the unimpaired development and function of reproductive tissues.
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In the first place, we wish to express our exceptional gratitude to Professor Freimut Leidenberger, the founder and constant supporter of the Institute for Hormone and Fertility Research, which in the more than 20 years of its existence provided an outstanding research environment, allowing us to make our contributions to the elucidation of the molecular mechanisms controlling orphan nuclear receptor functions. Furthermore, we thank Dr. Bert Vogelstein for the permission to use the AdEasy adenoviral vector system, Drs. T. Wieland and F. Schnieders for supplying the vector plasmids and the HEK 293 cells and their help in setting up the AdEasy system, Drs. K.I. Morohashi, K. Parker, J. Lund, and H.A. Ingraham for the gifts of the NR5A1 wild-type and mutant plasmids, Drs. L. Budnik and B. Brunswig-Spickenheier for their advice on bovine theca cell cultures, R. Domagalski and G. Tillmann for expert technical assistance, Dr. R. Soltau and his colleagues for the reliable supply with bovine ovarian tissues, and the NIADDK and the National Hormone and Pituitary Program for the gift of bovine LH. This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant no. WA 1152/2-1).
Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, D-20251, Hamburg, Germany
Norbert Walther, Martina Jansen, Wasima Akbary & Richard Ivell
School of Life Science Hamburg, University Hospital Eppendorf, Hamburg, Germany
Norbert Walther
Research Unit Molecular Oncology, Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany
Martina Jansen
Allergopharma Joachim Ganzer KG, Reinbek, Germany
Wasima Akbary
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
Richard Ivell
Correspondence to Norbert Walther.
NW conceived the study, constructed the adenoviral expression vectors, established and performed the quantification of transcripts by real time-PCR, and drafted the manuscript. MJ prepared and tested the adenovirus stocks and established the theca cell cultures for infection experiments. WA performed long-term cultures and adenovirus infection experiments. RI participated in the design of the study and the long-term cultures of differentiating theca cells.
Walther, N., Jansen, M., Akbary, W. et al. Differentiation-specific action of orphan nuclear receptor NR5A1 (SF-1): transcriptional regulation in luteinizing bovine theca cells. Reprod Biol Endocrinol 4, 64 (2006). https://doi.org/10.1186/1477-7827-4-64
Recombinant Adenovirus
Theca Cell
RAF1 Kinase
Orphan Nuclear Receptor


text:
"Fool for Your Loving" is a song recorded by British hard rock band Whitesnake. Originally released on their 1980 album Ready an' Willing, it was re-recorded for their 1989 album Slip of the Tongue.

Background and Recording
The song was Co-written by David Coverdale, Bernie Marsden and Micky Moody and was inspired by the breakup of Coverdale's first marriage. In Firecracker magazine, Coverdale said:

"You can look at my first marriage and see "Fool for Your Loving" and "Don't Break My Heart Again." A lot of those were fueled by songs about a relationship that once was very positive but sadly was unfolding into not positive." The song was originally written for blues legend B. B. King.

The song was the first big hit of Whitesnake's, reaching number 13 on the UK Singles Chart and number 53 on the U.S. Billboard Hot 100.
This remains one of Whitesnake's most popular and well-known songs. David Coverdale has stated that he prefers the original to the 1989-version.
A music video was also made, which features the band performing the song live on stage.

Re-recorded
As with the previous album, an old Whitesnake song was re-recorded for the band's 1989-album Slip of the Tongue. This time the band chose their 1980 UK-hit "Fool for Your Loving".
The re-recorded version was the first single released from Slip of the Tongue, but according to David Coverdale, the song "Judgement Day" was originally supposed to be the first single, but the record company insisted on "Fool for Your Loving". In the booklet of Slip of the Tongue 20th Anniversary Edition, David Coverdale comments :

"I was mortified when I allowed myself to be talked into letting Geffen release the re-recorded version of "Fool for Your Loving", instead of "Judgement Day" as the first one out of the box to promote the album... I knew radio would be all over "Judgement Day" just from the market research we did back then... but, Kalodner, Rosenblatt, Marco Babineau, my manager and some of our radio people, all people whose opinions I trusted, came down to the Record Plant when I was finishing off the album and all confronted me with what they felt was the way to go... that it would be a mistake to go with "Judgement Day"... Not only I but the band were really upset about that decision... I've regretted it ever since... I have no doubt it was Kalodner's idea, thinking we could achieve the same as we'd had with the re-recorded "Here I Go Again"... Anyway...they were wrong and so was I to go along with it... Another hard lesson learned... Stick to your guns if you believe in it...It's the only way..."

After its release, the re-recorded version charted  better in the US than the original, reaching number 37 on the Billboard Hot 100, number 2 on the Mainstream Rock Charts, but in the UK it failed to match the original's success, only reaching number 43.

A music video was also made for the song featuring the band performing on a stage, with smoke and headlights. There are also scenes of singer David Coverdale in a padded room and scenes of Coverdale's then-wife, Tawny Kitaen being chased by a car. Guitarist Adrian Vandenberg also appears in the video, even though he does not play on the song, due to a hand injury he sustained prior to the recording.

Track listing
1980
"Fool for Your Loving" - 4:18
"Mean Business" - 3:48
"Don't Mess with Me" - 3:25

1989
"Fool for Your Loving" - 4:10
"Slow Poke Music" - 3:59
"Walking in the Shadow of the Blues" (Live) - 4:49

Personnel
1980
David Coverdale – Vocals
Micky Moody – Guitars
Bernie Marsden – Guitars
Neil Murray – Bass
Ian Paice – Drums
Jon Lord – keyboards

1989
David Coverdale – Vocals
Steve Vai – Guitars
Adrian Vandenberg - Guitars (credited, but does not actually appear)
Rudy Sarzo – Bass
Tommy Aldridge – Drums

Chart performance

References

1980 songs
1980 singles
1989 singles
Whitesnake songs
Songs written by David Coverdale
Songs written by Bernie Marsden
Songs written by Micky Moody
Song recordings produced by Keith Olsen
Song recordings produced by Martin Birch
United Artists Records singles

redpajama_set_name: RedPajamaWikipedia


text:
Polo Grounds Baseball
Groundout Radio #86
54. Surfside Bar & Grill
Classification: Specialty
Location: Mound, Minnesota
I have thought about this more than most people, but I eventually reached the same conclusion as the majority: yes, flatbread is pizza. This is a variation on the "is a hot dog a sandwich?" meme* that has torn at the fabric of our nation for several years, and I can really see both sides of that particular argument. But there's not a really strong case to be made against including flatbreads in the writ large pizza category. Sub-category? I went with specialty here, because it remains slightly different.
* OK, the hot dog as sandwich debate has probably only torn at my family, but the fissures appear to be insurmountable at this point. The topic still comes up--long after the rest of the internets have moved on. But in a semi-related footnote, I have a (non-blood) relative that still argues, without common sense, merit or legal foothold, that M & Ms are a candy bar. Not a candy. A candy bar. Nobody argues that it's an excellent candy. But to consider it as a bar of any sort is unsupportable, and yet here I am, still belaboring the obvious after a decade (or more) of absurd debate. Still, let me know your thoughts.
Has the flatbread been created out of convenience, merely re-shaped to fit ovens and serving dishes, often at dining establishments with overly expansive offerings? I'll never view a flatbread as a "trendy" menu item, but part of me thinks they might have been borne out of the desire to appear more enlightened than serving up a lower-grade pizza. And I don't mean to malign flatbreads; no, quite the opposite. I almost always enjoy them when dining at restaurants typically featuring burgers, wings, and salads the size of a mixing bowl. During the early days of the pandemic, we enjoyed curbside service from Surfside, and the take-home was as satisfying as dining in front of the 84" screen while watching the Sabres-Maple Leafs game, which is our normal routine. Good flatbread, and not to overstate the obvious, but we need to get sports back quickly, because even hockey is sounding pretty decent right now. (7 of 10 stars)
1. Red's Savoy Inn
2. Mama's Pizza
3. The Brothers Three
© 2023 Polo Grounds Baseball Advanced Media. All rights reserved.

redpajama_set_name: RedPajamaCommonCrawl


OK, OK I was a little bit cruel to Balls Brothers in my Dion post, but do you know what? I went there this week and I was kind of right.
For the sake of clarity, I have never eaten at the restaurant part of a Balls Brothers, but I have eaten in the bar bit numerous times at numerous locations. Balls Brothers are like the McDonalds of the City. They are everywhere you look. There are nine locations, and within a five minute walk from the office I can count four.
All the locations I know are in basements, and they tend to be quite dark affairs. Burgundy walls and ceilings, wall lamps scattered throughout and framed Victorian style prints on the walls. The type of place where after emerging from lunch into the bright of day you kind of have to blink a few times. The small tables seemed to be scattered everywhere. You get the feeling they want you to think that the place is from older times.
Balls Brothers does attract an older crowd, but to be fair I thought the food was better than Dion even if the decor was not really my taste. Our group of three shared the cocktail sausages to start, then a selection of three sandwiches, some chips and some more sausages (which I think were mistakenly ordered). For the sake of comparison with my meal at Dion I ordered a smoked salmon sandwich. The bread was denser and the salmon better. The prices are also much more reasonable. All the sandwiches and light bites are between GBP5-10. I suppose they make their money on the wine, but that too is reasonably priced with bottles starting at GBP19.
The staff are polite, and service is fairly efficient. They are popular, and you'll find many of the tables reserved when you arrive. It's a solid choice for a wine bar, but there's just something a bit frumpy about the affair. I feel like I should be alot older, male and talking about my golf game on the weekend. But maybe that's just me.
Good For: A casual work lunch or just a casual glass of wine.
Just found your blog- am thoroughly enjoying plowing through the archives. Just relocated to London from Sydney- am loving having some wise recommendations on where to go!
Tori, I am so happy you are enjoying my blog! I am shortly moving to Montreal (middle of next month) but I still have a ton of restaurant reviews to post!


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text:
Place:Clinton, Indiana, United States
Robinsonbill
Name Clinton
Alt names Clinton source: Getty Vocabulary Program
Type County
Coordinates 40.267°N 86.5°W
Located in Indiana, United States (1830 - )
See also Delaware, Indiana, United States Parent county (source: Source:Population of States and Counties of the United States: 1790-1990)
Wabash, Indiana, United States Parent county (source: Source:Population of States and Counties of the United States: 1790-1990)
Contained Places
Inhabited place
Boyleston
Edna Mills
Geetingsville
Greetingville
Hillisburg
Kirklin
Mattix Corner
Michigantown
Middlefork
Scircleville
Wesley Manor
Woodside Park
Clinton County is a county located in the U.S. state of Indiana. As of 2010, the population was 33,224. The county seat is Frankfort.
3 Population History
4 Cemeteries
5 Research Tips
Clinton County officially came into existence on March 1, 1830, and was named in honor of DeWitt Clinton, the seventh Governor of New York State and architect of the Erie Canal, which opened up the Upper Midwest to settlement. The act forming the county was approved by the Indiana General Assembly on January 29, 1830, and created Clinton from the eastern parts of neighboring Tippecanoe County.
Lieutenant General James F. Record was born and raised in Clinton County; Gen Record was awarded 3x Distinguished Service Crosses for his gallantry during the Vietnam War.
1829 Land records recorded Source:Red Book: American State, County, and Town Sources
1830 County formed Source:Red Book: American State, County, and Town Sources
1830 First census Source:Population of States and Counties of the United States: 1790-1990
1830 Marriage records recorded Source:Red Book: American State, County, and Town Sources
1830 Probate records recorded Source:Red Book: American State, County, and Town Sources
1832 Court records recorded Source:Red Book: American State, County, and Town Sources
1860 No significant boundary changes after this year Source:Population of States and Counties of the United States: 1790-1990
1882 Birth records recorded Source:Red Book: American State, County, and Town Sources
Population History
source: Source:Population of States and Counties of the United States: 1790-1990
Census Year
Cemeteries of Clinton County, Indiana, United States
Clinton County INGenWeb
This page uses content from the English Wikipedia. The original content was at Clinton County, Indiana. The list of authors can be seen in the page history. As with WeRelate, the content of Wikipedia is available under the Creative Commons Attribution/Share-Alike License.
Retrieved from "https://www.werelate.org/wiki/Place:Clinton%2C_Indiana%2C_United_States"
Category: Clinton, Indiana, United States
This page was last modified 19:01, 9 March 2015.


A Pioneer in Heat Pump Technology.
The world is in a transition from fossil fuels to clean efficient energy. Innovative companies accelerate this process by providing mature alternatives, for the benefit of the environment and future generations, to benefit all people that have a vision for a sustainable future. An important factor is heating.
Team spirit is moving towards one and the same!
A true impression is that Heliotherm heat pumps has set its place in the future, having made their signature mark in the present. More than 35.000 Heliotherm heat pumps have been installed throughout Europe. Their efficient performance, economical function and reliability are based on the state-of-the-art technology concept. Also, our dependable 85 employees.
Heliotherm is a Technology Leader in Europe.
Our invitation is to all who want to get out the unforeseeable world of fossil fuels: Move into Independence! - with a Heliotherm heat pump.
The achievement of the ISO 9001:2008 certification confirms the European heat pump innovation leader based in Tyrol of its first-rate quality management.
During the certification audit, the practical application and effectiveness of the quality management system at Heliotherm was reviewed and evaluated. In accordance to the ISO 9001:2008 criteria observance, which guarantees maximum performance and work efficiency. The most important principles are: strong customer orientation, process and solution-oriented decision making and continuous optimization measures.
The independent and external certification office ALL-CERT has concluded that Heliotherm fulfills all requirements without hindrance and fulfills them exemplary. The certification is valid for three years and the included annual monitoring audits ensure that the quality management system is lived, maintained and continuously improved. Heliotherm Managing Director Andreas Bangheri: since it was not mandatory to comply with this certification; the completion compliments our highest quality standards. It was simply important to us to confirm this high standard through an objective authority.
The promotion agency Tirol is an institution with the legal mandate to sustainably strengthen the economic and scientific location of Tyrol; thereby securing existing jobs and creating new ones. The cluster initiatives are co-financed by the ERDF (European Regional Development Fund).
EHPA represents the interests of the European heat pump industry on an international level. Heat pumps contribute to the achievement of European climate protection targets and are already standard solutions in Sweden and Switzerland.
This initiative creates regulations for the definition and implementation of an energy efficiency label for heat pumps. The energy efficiency class of the model is given by a scale from ++ (very efficient) to -- (less efficient).
klima:aktiv is the initiative launched by the Lebensministerium (Ministry of Life) for active climate protection and part of the Austrian climate strategy launched in 2004. The aim is the rapid and broad market introduction of climate-friendly technologies and services.
The environmental arena makes sustainability possible and explains ideas and technologies from different sectors and areas of everyday life. Visitors can explore the combined knowledge, compare and grasp solutions and draw a concrete benefit from everyday life.
The marketplace works towards increasing the popularity of the heat pump and the geothermal study provides potential consumers with planning assurance when they want to use their garden as a source of ground energy.
BBA MCS: BBA (British Board of Agrément), is an independent testing and certification service that supports manufacturers and installers achieve excellence and provide reassurance throughout the industry. The Microgeneration Certification Scheme (MCS) certififies renewable energy technologies and Microgeneration products, in accordance with documented standards.
The BWP represents the interests of the entire heat pump industry. The focus of the BWP is the improvement of general conditions, PR and public relations regarding heat pumps, networking and exchange at specialist events, quality assurance and heat pump certification; the Seal of Quality, drilling companies and heat pump installers.
The KlimaHaus Agency is an institution of the Italian Province of Bolzano for the energy certification of buildings as well as for education and training for all involved in construction. The agency has certified over 5,000 buildings and organized courses for more than 22,000 planners. The Agency is also organizing initiatives to raise public awareness of energy efficiency, sustainability and climate change. The KlimaHaus agency sensitizes, informs and promotes transparency to further energy-efficient and sustainable construction.
The Association Wärmepumpe Austria (WPA), exists since 2012, covers the industry's entire value chain. Its main tasks include the unification and strengthening of the industry, the creation of a better framework for heat pumps and the quality assurance of the entire heat pump system.
The Fair Energy Partners are committed to the responsibility to the use of energy: Sustainable technologies and high quality standards offer potential for optimization for households and farms as well as for industry, industry and municipal facilities. And this potential is achieved by means of modern, contemporary solutions: because we perceive this to be fair to future generations.
We increasingly support young athletes and regional clubs in the Kufstein area. Whether it is soccer, racing, handicapped athletes or children with disabilities - we are happy to promote them to make a valuable contribution in our society.
Amongst these successful athletes and as brand ambassador with Heliotherm Wärmepumpentechnik GmbH is the successful Austrian and international sled racer Reinhard Egger with his manager Tobias Schiegl.
You are welcome to send us your sponsoring request.


Mariusz Duda is now presenting the follow-up of Fractured, his last years Lunactic Soul release. Under The Fragmented Sky was recorded at the same time, same studio. It's like the next chapter.
The album opens with He av En, and it's an unusual opening. Two voices singing, but no words, accompanied by a tone. Monotone at first the tune develops into an intense song. A bit confusing. But then Trials kicks off with a heavy humming sound, electronic sounds and drums join in - and finally Mariusz Duda starts singing. It's still kinda moody, like Fractured, but with the repetitiveness of the new tracks it gets a different feel. It's still dark and moody, but also lighter. Even is Trials is almost threatening towards the end. A guitar dominates Sorrow, a short instrumental, an interlude. At Under The Fragmented Sky the touching vocals are back and so connect this release with the predecessor.
Rinsing The Night has an instrumental with an Oriental touch, woven into the thick bass sound and synth sounds. The Art Of Repairing seems to skip and is a bit strange... somehow make me think of Kraftwerk... But while keeping the opening theme, Mariusz Duda adds some Tangerine Dream like sounds and creates a new soundscape.
The closer Untamed could have been also the closer of Fractured, it's mainly acoustic guitar and vocals.
At this tune Duda welcomes Wawrzyniec Dramowicz (aka Vaaver) on drums. Everything else on this mini-album is by Mariusz Duda.
While Fractured is easily accessible for the listener, Under The Fragmented Sky can be quite challenging. Lunactic Soul can be everything, it's simply Duda's musical vision at that time of his life. Let's see what he'll have for his fans next time!
Mariusz Duda, known as the mastermind of Riverside, founded Lunatic Soul to have an outlet for his creativity. Now he presents Fractured. In the case of Fractured he had to overcome a personal tragedy. He kinda came back to life after what happened in 2016 and this is his way to cope with it. And so it's no surprise that the songs have a dark edge.... moody feeling.
He kicks off with Blood On The Tightrope, a progressive tune with electronic elements. But it's Duda's voice who gives the song it's character. Towards the end the bass line is picking up speed and starts to dominate the tune. It's like something threatening is coming closer - and than it's gone.
Anymore is dominated by electronic sounds and the fat bass line as a counterpart. It's like a fight, an inner fight of Mariusz Duda following the tragedies.
The songs are somehow minimalistic, even if here and there he adds something to the basics - like the strings at Crumbling Teeth And The Owl Eye and saxophone played by Marcin Odyniec at Red Light Escape - and overall it's his vocals, his lyrics which make this album so intense. The title track is picking up speed and offers some almost hypnotic sound elements.
With A Thousand Shards Of Heaven he presents the longest track, a slow tune with acoustic guitar and strings by Poland's Sinfonietta Consonus Orchestra. The song is full of sadness, but also carries a spark of hope. In the middle of this opus he picks up speed and ends in a mid-tempo passage dominated by Duda's bass accompanied by saxophone occasionally.
Duda is focusing this time on moods, his vocals and enriches the musical journey with electronic sounds and sparse instrumentation. But also surprises the listener every now and then.


text:
Saintel Daily
If it Happened | We Covered it
Huawei Forced to Leave Google
When you think about any one of the tech giants, I assume, like me, you are of the impression that they have gotten too big. Now, don't get me wrong, I benefit from a lot of these services, and dare I say it – they make my life easier. But at what point are they too big? Now, this argument has been made for years and is being made by lawmakers right now in an attempt to get support for the upcoming election. Outside of regulation, is there anything that can be done to stop the likes of Google, for example?
According to Huawei, there is. In fact, the Chinese tech giant is gearing up to take on Google Maps. Huawei has started developing its own mapping service known as Map Kit. But there's a catch – this isn't a solution for consumers, but rather, Huawei wants to give developer tools to build new applications based on its mapping capabilities. This is an interesting idea, but will it work?
The service will be available in 150 countries and regions, will be connected to local mapping services. It will also support 40 different languages. It's being rumored that it will also include real-time traffic conditions, sophisticated navigation capabilities and augmented reality mapping. In addition to this, Huawei has already gotten Yandex on board. If you're not familiar with Yandex, they are commonly referred to as the Russian Google. Huawei has also gotten Booking.com on board. It seems that others have gotten tired of Google being such a behemoth, and are supporting Huawei in this venture.
That said, this news comes only days after Huawei confirmed rumors that they are building their own operating system for mobile devices, laptops, wearables, and televisions. The new operating system will be known as Harmony OS. And this was sparked after Google banned Huawei from using their Android OS after the Chinese manufacturer ended up on the US entity list. Since then, Huawei has been looking into alternatives in order to decrease its reliance on Google and its services. Which means if Huawei wants to stay in business, they have no choice but to adapt.
Regardless, this is all very interesting. I mean, Huawei became public enemy number one in the United States, which meant that Google had to put a stop to any kind of involvement they had with the Chinese company. Otherwise, their products would have been at risk as well. I say this is interesting because I don't believe that the information against Huawei has been substantiated. And we all know that Donald Trump is in the middle of a trade war with China. I mean, you can definitely put two and two together.
That said, it was only a matter of time before another company came out with an operating system. Sure, it might not last, and it might not make it to North America, but it will further divide the United States from China and maybe other parts of the world. Even though it seems like Huawei is taking on Google, their actions are really as a result of something Google did to them in the first place. Next time you hear something, make sure you dig a little deeper to get a better understanding of the rest of the story.
Posted in TechnologyTagged Android, Google, HarmonyOS, Huawei, TechnologyBy Staff Writer1 Comment
One thought on "Huawei Forced to Leave Google"
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Richard Brent is an Emeritus Professor at the Australian National University and Conjoint Professor at the University of Newcastle.
He was appointed Foundation Professor of Computer Science at the Australian National University in 1978.
In March 1998 he became the Statutory Professor of Computing Science in the University of Oxford.
In March 2005 he took up a 5-year position as ARC Federation Fellow in the Mathematical Sciences Institute and the Research School of Information Sciences and Engineering at the Australian National University.
He was also Visiting Professor at the University of Oxford Sept 2005 - Sept 2011.
In March 2010 he became a Distinguished Professor of Mathematics and Computer Science with a joint appointment in the Mathematical Sciences Institute and the Research School of Computer Science at the Australian National University. Since September 2011 he has been an Emeritus Professor.
Chief Investigator with A. Guttmann et al in the ARC Centre of Excellence for Mathematics and Statistics of Complex Systems (MASCOS) CE0348217, $17M for 2003-2010.
Chief Investigator with J. Cannon and D. Stehlé in the ARC Discovery grant DP0880724 Integral lattices and their theta series, $360K for 2008-2010.
Chief Investigator with B. B. Zhou in the ARC Discovery grant DP0985271 An integrative and interactive approach for co-estimation of multiple sequence alignment and phylogeny reconstruction, $230K for 2009-2011.
Chief Investigator with M. Hegland et al in the ARC Linkage Project LP110200410 Robust numerical solution of partial differential equations on petascale computer systems with applications to tsunami modelling and plasma physics, $446K for 2011-2014.
Chief Investigator with J. Borwein and D. Bailey in the ARC Discovery Grant DP140101417 Exploratory Experimentation and Computation in the Mathematical Sciences: Theory and Practice, $415K for 2014-2017. The rationale is given here.
Moyal Medal, Macquarie University, 2014.
A short cv (updated August 2018) is available in pdf format. A summary of research (updated 2016) is also available in pdf.


text:
Home > The Agency > Awards > AFP rewarded at World Press Photo 2018
AFP rewarded at World Press Photo 2018
The 2018 edition of the World Press Photo has rewarded three AFP photojournalists, including Ronaldo Schemidt, one of six finalists for the World Press Photo of the Year.
Ronaldo Schemidt's shot of a protester on fire during clashes with police in Venezuela was selected by the jury as one of the six best photos of the year. He was also nominated in the "Spot Singles" category with the same photo. Ronaldo Schemidt, a Venezuelan national, started working with AFP in 2004; he joined the Mexico City bureau in 2006 and is now based there. He has covered a number of events, including the Copa America in Argentina, World Cup in Brazil and the struggle against drug traffickers in Mexico.
Juan Barreto was also rewarded for his coverage of the Venezuelan conflict. His series, featuring the same protester on fire, is one of the three shortlisted in the "Spot Stories" category. The Venezuelan photographer joined the Agency in 1999 and covers current affairs in the region: political crises, presidential elections, the earthquake in Haiti, sporting events, etc. He is based in the Caracas bureau and covers the Venezuelan crisis continuously.
Oli Scarff was nominated in the "Sport Singles" category for a photograph taken during the "Royal Shrovetide football" match, a traditional blend of football and rugby played once a year by the people of Ashbourne, a village in central England. Based in the Manchester area, Oli Scarff joined AFP in 2014 after working for the Daily Telegraph and then Getty Images. He covers the news in the north of England.