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The cell cycle depends on a sequence of steps that are triggered and terminated via the synthesis and degradation of phase-specific transcripts and proteins. Although much is known about how stage-specific transcription is activated, less is understood about how inappropriate gene expression is suppressed. Here, we demonstrate that Groucho, the Drosophila orthologue of TLE1 and other related human transcriptional corepressors, regulates normal cell cycle progression in vivo. We show that, although Groucho is expressed throughout the cell cycle, its activity is selectively inactivated by phosphorylation, except in S phase when it negatively regulates E2F1. Constitutive Groucho activity, as well as its depletion and the consequent derepression of e2f1, cause cell cycle phenotypes. Our results suggest that Cdk1 contributes to phase-specific phosphorylation of Groucho in vivo. We propose that Groucho and its orthologues play a role in the metazoan cell cycle that may explain the links between TLE corepressors and several types of human cancer., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition., Competing Interests: The authors have declared that no competing interests exist.

Gene expression is often controlled by transcriptional repressors during development. Many transcription factors lack intrinsic repressive activity but recruit co-factors that inhibit productive transcription. Here we discuss new insights and models for repression mediated by the Groucho/Transducin-Like Enhancer of split (Gro/TLE) family of co-repressor proteins.

Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in "active" chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and polymerase pausing.

The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. Here we demonstrate that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. Our results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner.

Factors affecting transcriptional elongation have been characterized extensively in in vitro, single cell (yeast) and cell culture systems; however, data from the context of multicellular organisms has been relatively scarce. While studies in homogeneous cell populations have been highly informative about the underlying molecular mechanisms and prevalence of polymerase pausing, they do not reveal the biological impact of perturbing this regulation in an animal. The core components regulating pausing are expressed in all animal cells and are recruited to the majority of genes, however, disrupting their function often results in discrete phenotypic effects. Mutations in genes encoding key regulators of transcriptional pausing have been recovered from several genetic screens for specific phenotypes or interactions with specific factors in mice, zebrafish and flies. Analysis of these mutations has revealed that control of transcriptional pausing is critical for a diverse range of biological pathways essential for animal development and survival., (© 2013 WILEY Periodicals, Inc.)

One of the many advantages of Drosophila melanogaster as a model organism is the relative ease with which gene deletions can be generated by imprecise excision of transposon insertions. Here, we describe a simple, fast, and efficient method of screening for single-gene excision events that is not biased by prior assumptions of the mutant phenotype. DNA sequence polymorphisms were used as co-dominant electrophoretic markers to identify candidate deletions in a single generation, and to delimit the breakpoints to within 0.5-1 kb, thereby rapidly identifying deficiencies that affect only the gene of interest. In addition, we used polymorphism profiling to map existing deficiencies. The method can also be applied to map the extent of deletions generated by x-rays and to identify targeted mutations generated by engineered zinc-finger nucleases in Drosophila and other polymorphic model organisms (e.g., zebrafish, mouse, Caenorhabditis elegans).

Bio-electrosprays (BESs) provide a means of precisely manipulating cells and thus have the potential for many clinical uses such as the generation of artificial tissuesorgans. Previously we tested the biological safety of this technology with a variety of living cells and also embryos from the vertebrate model organisms Danio rerio (zebrafish) and Xenopus tropicalis (frog). However, the viability and fertility of the treated embryos could not be fully assessed due to animal licensing laws. Here we assay the viability and fertility of Drosophila melanogaster (fruit fly) embryos in conjunction with the bio-electrospray procedure. Bio-electrosprayed Drosophila embryos developed into fully fertile adult flies that were indistinguishable from wild-type. Thus, we demonstrate that the bio-electrospray procedure does not induce genetic or physical damage that significantly affects the development or fertility of a multicellular organism. This study along with our previous investigations demonstrates the potential of this approach to be developed for the precise manipulation of sensitive biological materials.

Rice bran oil (RBO) was enzymatically modified in a continuous packed bed bioreactor to incorporate caprylic acid with Lipozyme RM IM as biocatalyst. The reaction product was purified by short-path distillation. Rice bran oil structured lipid (RBOSL) contained 32.1 mol % caprylic acid. Positional analysis revealed 0.7 mol % caprylic acid at the sn-2 position and 47.8 mol % caprylic acid at the sn-1,3 positions. Composition of free fatty acids and smoke point of RBO and RBOSL were not significantly different. Saponification value, iodine value, and viscosity of RBO were significantly different from those of RBOSL. The color of RBOSL was darker, more yellow and less green than RBO. Volatile compounds in RBO and RBOSL were determined by GC-MS. Melting onset temperatures of RBO and RBOSL were not significantly different, while melting end point temperatures and melting enthalpies were significantly different. This characterization study results will help determine potential food applications of RBOSL.

The Drosophila Groucho (Gro) protein was the founding member of the family of transcriptional co-repressor proteins that now includes the transducin-like enhancer of split (TLE) and Grorelated gene (Grg) proteins in vertebrates. Gro family proteins do not bind DNA directly, but are recruited by a diverse profile of transcription factors, including members of the Hes, Runx, Nkx, LEF1/Tcf, Pax, Six and c-Myc families. The primary structure of Gro proteins includes five identifiable regions, of which the most highly conserved are the amino-terminal glutamine-rich Q domain and the carboxy-terminal WD-repeat domain. The Q domain contains two coiled-coil motifs that facilitate oligomerization into tetramers and binding to some transcription factors. The WD domain folds to form a beta-propeller, which mediates protein-protein interactions. Many transcription factors interact with the WD domain via a short peptide motif that falls into either of two classes: WRPW and related tetrapeptides; and the 'eh1' motif (FxIxxIL). Gro family proteins are broadly expressed during development and in the adult. They have essential functions in many developmental pathways (including Notch and Wnt signaling) and are implicated in the pathogenesis of some cancers. The molecular mechanisms through which Gro proteins act to repress transcription are not yet well understood. It is becoming clear that Gro proteins have different modes of action in vivo dependent on biological context and these include direct and indirect modification of chromatin structure at target genes.

The Groucho (Gro)/transducin-like enhancer of split family of transcriptional corepressors are implicated in many signalling pathways that are important in development and disease, including those mediated by Notch, Wnt and Hedgehog. Here, we describe a genetic screen in Drosophila that yielded 50 new gro alleles, including the first protein-null allele, and has two mutations in the conserved Q oligomerization domain that have been proposed to have an essential role in corepressor activity. One of these latter mutations, encoding an amino-terminal protein truncation that lacks part of the Q domain, abolishes oligomerization in vitro and renders the protein unstable in vivo. Nevertheless, the mutation is not a null: maternal mutant embryos have intermediate segmentation phenotypes and relatively normal terminal patterning suggesting that the mutant protein retains partial corepressor activity. Our results show that homo-oligomerization of Gro is not obligatory for its action in vivo, and that Gro represses transcription through more than one molecular mechanism.

The Groucho (Gro)/TLE/Grg family of corepressors operates in many signaling pathways (including Notch and Wnt). Gro/TLE proteins recognize a wide range of transcriptional repressors by binding to divergent short peptide sequences, including a C-terminal WRPW/Y motif (Hairy/Hes/Runx) and internal eh1 motifs (FxIxxIL; Engrailed/Goosecoid/Pax/Nkx). Here, we identify several missense mutations in Drosophila Gro, which demonstrate peptide binding to the central pore of the WD (WD40) beta propeller domain in vitro and in vivo. We define these interactions at the molecular level with crystal structures of the WD domain of human TLE1 bound to either WRPW or eh1 peptides. The two distinct peptide motifs adopt markedly different bound conformations but occupy overlapping sites across the central pore of the beta propeller. Our structural and functional analysis explains the rigid conservation of the WRPW motif, the sequence flexibility of eh1 motifs, and other aspects of repressor recognition by Gro in vivo.

Segmental patterning in Drosophila relies on a cascade of transcription factors that subdivide the embryo into successively more precise domains. We have identified a missense mutation (W049) in the gene encoding the transcriptional elongation factor Spt5 (reviewed in ) which, when homozygous in the maternal germ line, leads to defects in segmental patterning of the embryo. W049 alters the C-terminal domain of Spt5 and affects its activity in vitro, impairing its abilities to confer sensitivity to the transcriptional inhibitor DRB and to stimulate transcription at limiting nucleotide concentrations. In vivo, W049 shows locus-specific effects on transcription: expression of gap genes remains wild-type, but striped patterning of the primary pair-rule genes even-skipped and runt is disrupted. even-skipped stripes are broadened in the mutant embryos indicating that Spt5 is likely to be a direct, negative regulator of this target gene. Our results suggest control of transcriptional elongation by repressors contributes to striped gene expression in the embryo. By contrast, expression of heat shock-induced proteins is reduced in the mutant embryos. These results provide genetic evidence for Spt5 function during heat shock induction and demonstrate that Spt5 acts both positively and negatively on transcription in vivo depending on context.

Capric acid (C10:0) was incorporated into rice bran oil with an immobilized lipase from Rhizomucor miehei as the biocatalyst. Effects of incubation time, substrate mole ratio, enzyme load, and water addition on mole percent incorporation of C10:0 were studied. Transesterification was performed in an organic solvent, hexane, and under solvent-free condition. Pancreatic lipase-catalyzed sn-2 positional analysis and tocopherol analysis were performed before and after enzymatic modification. Products were analyzed by gas-liquid chromatography (GLC) for fatty acid composition. After 24 h of incubation in hexane, there was an average of 26.5 +/- 1.8 mol % incorporation of C10:0 into rice bran oil. The solvent-free reaction produced an average of 24.5 +/- 3.7 mol % capric acid. In general, as the enzyme load, substrate mole ratio, and incubation time increased, the mole percent of capric acid incorporation also increased. Time course reaction indicated C10:0 incorporation increased up to 27.0 mol % at 72 h, for the reaction in hexane, and up to 29.6 mol % at 12 h, for the solvent-free reaction. The highest C10:0 incorporations (53.1 and 43.2 mol %) for the mole ratio experiment occurred at a mole ratio of 1:8 for solvent and solvent-free reactions, respectively. The highest C10:0 incorporation (27.9 mol %) for the reaction in hexane occurred at 10% enzyme load, and the highest incorporation (34.4 mol %) for the solvent-free reaction occurred at 20% enzyme load. Incorporation of C10:0 into rice bran oil declined with the addition of increasing amounts of water after reaching 30.3 mol % at 2% water addition in hexane, and in the solvent-free reaction after reaching 35.9 mol %.

Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.

In a randomized, double-blind crossover study, the effects of 0.8, 1.6 and 3.2 mg/day inhaled budesonide and 5, 10 and 20 mg/day oral prednisolone on mineral metabolism were compared. Twelve healthy subjects (4 m, 8 f) were treated for 1 week at each dosage level, the graduated dosages being given in ascending order. Budesonide and prednisolone were given twice daily and once daily, respectively, which reflects the schedules common in clinical practice. Serum calcium and the regulatory hormones of calcium metabolism (parathyroid hormone, vitamin D metabolites and calcitonin) were not changed either by prednisolone or budesonide. Prednisolone significantly increased 24 h and 08.00 h fasting urinary calcium excretion and decreased renal calcium reabsorption, while budesonide had little or no effect on urinary calcium loss and increased renal reabsorption at the highest dose level. Both drugs significantly increased renal phosphate reabsorption and serum phosphate levels, but prednisolone caused greater increases than budesonide. In conclusion, during short-term treatment with the dosages used, inhaled budesonide had less effect on calcium and phosphate metabolism than oral prednisolone, and so it may have a lesser action on the skeleton of the type contributing to osteoporosis during long-term treatment.

The effects of inhaled budesonide (BUD) and oral prednisolone (PRED) on markers of bone turnover and adrenal function were compared in a randomized, double-blind, double-dummy, crossover study. Twelve healthy subjects were treated for one week with 0.8, 1.6 and 3.2 mg/day BUD and 5, 10 and 20 mg/day PRED, the three doses being given in ascending order. Plasma cortisol and adrenal cortical androgens showed a significantly decreasing trend with the increasing doses of both drugs, although PRED caused a significantly greater decrease than BUD. Osteoblast function, reflected by serum osteocalcin and alkaline phosphatase was significantly reduced by PRED, but BUD had a significantly different effect as it affected only osteocalcin. Urinary hydroxyproline/creatinine, a marker of bone resorption, was not changed by either drug. The average potency ratio for equivalent systemic effects was PRED:BUD 3.9:1. During short-term treatment at equivalent anti-asthmatic doses, BUD has significantly less effect on adrenal function and bone turnover than PRED, and it may carry less risk of bone complications during long-term treatment.

Inhaled antiasthmatic steroids have been assumed and yet never proved to exert their antiasthmatic effect by topical action in the airways. We tested the hypothesis that the efficacy of inhaled budesonide (BUD) might be due instead to its systemic activity after absorption. We compared inhaled and oral BUD with doses selected to ensure higher peak plasma levels and a greater area under the plasma concentration curve with the oral treatment. After pretreatment with beclomethasone to maximize asthma control, 47 adults with asthma were randomized to receive 0.4 mg of inhaled BUD per day (n = 16) or 1.4 mg of oral BUD per day (n = 15), or placebo (n = 16) in double-blind fashion and then followed weekly until asthma relapsed or for 8 weeks if no relapse occurred. "Relapse" was defined as a drop in the mean peak expiratory flow rate greater than 2 SEM below the mean during the baseline week before switching to the test drugs. The time to relapse was the primary outcome variable. Time to relapse was longer with inhaled than with oral BUD (medians, 22 versus 7.9 days; p = 0.003) or placebo (medians, 22 versus 9 days; p = 0.004). Oral BUD and placebo did not differ (p = 0.41). The morning serum cortisol levels remained normal during all three treatments. Thus, at conventional dosage the antiasthmatic effect of inhaled BUD may be fully explained by a local intrapulmonary action.

Twelve healthy males (mean age 27.6 y, range 23-35 y) took part in a randomized, double-blind, cross-over study of the effect of blood sampling technique (separate isolated venepunctures vs use of an IV cannula) and frequency (overnight vs morning) on plasma cortisol and white blood cell count after inhalation of a single dose of budesonide 3.2 mg or placebo, in order to establish the more sensitive method for future use. Sampling technique and frequency affected neither leucocytes nor plasma or urinary cortisol. Budesonide suppressed both plasma and urine free cortisol and delayed the nocturnal rise due to the circadian rhythm, thus reducing the AUC of plasma cortisol vs time. Lymphocytes, eosinophils and monocytes were decreased and neutrophils and total white blood cells were increased by the high dose of budesonide used. Lymphocytes and neutrophils showed significant changes earlier than eosinophils and cortisol and may be the variables of choice under certain conditions. Frequent sampling gave more complete information about the systemic effect of the drug than single morning samples.

Fifty-three adult asthmatic subjects requiring 800 micrograms or more of inhaled beclomethasone dipropionate were enrolled in a double-blind parallel group study of 6-month duration to compare the efficacy and side effects of inhaled budesonide in doses of 800, 1,200, and 1,600 micrograms given two or four times a day (BID or QID). After a two-week observation period to establish baseline, subjects were given the same dose of budesonide as they would be for beclomethasone dipropionate; however, the frequency regimen (BID or QID) was randomly allocated. Subjects were asked to fill in diary cards describing their asthmatic symptoms, need for medication, and throat symptoms. They were assessed by a physician every 4 wk, at which time spirometry was performed. Throat swabs were done at every other monthly visit. Cortisol and response to cortisol after synthetic ACTH injection were assessed at the beginning and end of the trial. Thirty-six subjects, half in each group, completed the study. Those on the BID regimen had almost twice as many days with nocturnal asthma and cough and almost three times as many days with disability due to asthma. There were also twice as many relapses in the BID regimen as judged by the clinician. These relapses mainly occurred toward the end of the study. The maximal daily swings in peak expiratory flow rates were slightly greater in the BID group, although this difference was not physiologically significant. Spirometry, cortisol secretion, and the response after synthetic ACTH injection were not significantly different in either group from the beginning to the end of the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Four patients with pigeon breeder's lung and positive lymphocyte transformation tests to pigeon antigen were given 250 mg hydrocortisone intravenously, after which the test was repeated at 6, 24, and 30 hr. A significant reduction in lymphocyte responsiveness to pigeon antigen was evident 6 hr after the steroid dose, which reversed within 24 hr after the treatment. This was associated with transient 'false negative' lymphocyte transformation tests in two of the four patients, so this effect of steroid treatment may influence the reliability of this test, if used in the diagnosis of patients with allergic alveolitis. Phytohaemagglutinin responsiveness was not affected by the hydrocortisone treatment. This may indicate difference between the cell populations that respond, after steroid treatment, to antigen and to mitogenic stimulation.

4beta,5-Epoxy-5beta-androstane-3,17-dione (1a), 17beta-hydroxy-4beta,5-epoxy-5beta-androstan-3-one (1b) and 17beta-acetoxy-4beta,5-epoxy-5beta-androstan-3-one (1c) were treated with anhydrous hydrogen fluoride in pyridine (70% solution) at 55 detrees and yielded the corresponding 4-en-4-ols, e.g. 4-hydroxy-4-androstene-3,17-dione (2a). As the reaction temperature was lowered each epoxide formed a second product which, at -75 degrees, was the major component of the reaction mixture and was identified as the 5alpha-fluoro-4alpha-ol derivative of the parent enone, e.g. 4alpha-hydroxy-5-fluoro-5alpha-androstane-3,17-dione (3a). These fluorohydrins are thermally unstable, losing hydrogen fluoride. The acetates of -he fluorohydrins were also prepared, characterized, and shown to be more stable than the parent alcohols.

The compound 3 beta-acetoxy-5, 6 beta-dichloromethylene-5 beta-androstan-17-one crystallizes in the orthorhombic space group, P212121, with four molecules per unit cell, having the following dimensions: a = 7.936(2) A, b = 11.593(2) A, and c = 22.955(4) A. X-ray analysis of single crystal data (1764 observed reflections), collected on a Syntex P1 autodiffractometer, led to complete structural assignment. Solution by direct methods and refinement by least-squares calculations resulted in a final R of 0.037. The conformation of ring A represents one of the few examples of a distorted boat and ring B approximates a distorted chair, both conformations attributable to the strain of the cyclopropyl ring. The spatial relationship between the endo chlorine atom, and the angular methyl group, C(19) is 3.095(5) A. Ring C is a normal chair and ring D adopts a 13 beta-envelope conformation. The steroid molecules pack perpendicular to the c axis with screw axes intersecting the A-B ring junction.

A comparative study was made of the reactions of 5-bromo-3 beta, 6 beta-dihydroxy-5 alpha-androstan-17-one 3-acetate (1) with lead tetraacetate alone and in the presence of iodine in both high intensity visible light and in total darkness using a variety of solvents. Markedly different product profiles were obtained under the different reaction conditions, making our results of both practical importance and theoretical interest.