An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.     

Characteristics of consultants who hold distinction awards in England and Wales: database analysis with particular reference to sex and ethnicity

Objective To determine whether women, ethnic minorities, and particular specialties are discriminated against in the receipt of NHS distinction awards.Design Analysis of database of consultants eligible for distinction awards.Setting England and Wales, 2002.Main outcome measures Holding of B, A, and Aplus distinction awards, analysed for all awards, irrespective of when made, and for awards made in the last five years studied.Results Women and doctors from ethnic minorities were substantially under-represented among award holders when no account was taken of potential confounding factors. Differences diminished after multivariate analysis, but some remained significant. For example, the adjusted odds ratio of women holding awards compared with men was 0.69 (95% confidence interval 0.59 to 0.82) for any award and 1.37 (0.86 to 2.20) for Aplus awards; the odds ratio for any award for non-white doctors trained abroad compared with white doctors trained in the United Kingdom was 0.45 (0.37 to 0.56). In the last five years studied the adjusted ratio of women to men was 0.94 (0.79 to 1.10) for B awards and 1.54 (0.85 to 2.83) for Aplus awards. The adjusted ratio for non-white British trained consultants was 0.86 (0.62 to 1.17) for B awards and 1.20 (0.37 to 3.87) for Aplus awards; for non-white consultants trained abroad it was 0.68 (0.54 to 0.85) for B awards and 0.69 (0.15 to 3.10) for Aplus awards; and for white consultants trained abroad it was 0.70 (0.54 to 0.91) for B awards and 0.90 (0.38 to 2.15) for Aplus awards.Conclusion Historical under-representation in award holding by women and doctors from ethnic minorities was partly explained by time spent as a consultant. Recent awards showed no under-representation of women and no appreciable under-representation of ethnic minorities overall. However, doctors who trained abroad—both white and non-white—remained under-represented for B awards.     

Parentage and host preference in the common cuckoo Cuculus canorus

Microsatellite DNA markers were used to investigate parentage relationships in a population of common cuckoo Cuculus canorus. Thirty adults and 55 nestlings were genotyped at six loci from blood samples collected over a four‐year period. To test whether each cuckoo female specialises in parasitising one single host species (Host Preference Hypothesis), the maternal relationships were used to record each female's host choice. The results supported the Host Preference Hypothesis since no female (N=3) was recorded to have parasitised more than one of four congeneric host species breeding in the area. In contrast, the males (N=4) did not show such specialisation since two of them sired offspring reared by different host species.     

Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2-10 tandem repeat sequence copies (mean=4.82 copies) while ALS6 alleles had 2-8 copies (mean=4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5' of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.     

Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.     

Comments on the sub-group reports of the EU Technical Expert Working Group on the revision of Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes

A critical analysis is presented of the reports produced by four Technical Expert Working Group Sub-groups (SGs) on Ethical Review, Cost-Benefit, Authorisation and Scope, which were published on the EC website (http://ec.europa.eu/environment/chemicals/lab_animals/ia_info_en.htm), as part of the European Commission (EC)s review of EU Directive 86/609 EEC. This is in addition to our official response to the internet consultation questionnaire, submitted to the Commission on behalf of FRAME. Whilst the respective SG reports were extensive and detailed, we have identified several shortcomings, and in particular, a frequent lack of consensus among the SG members, resulting in a lack of clear guidance for the EC on what the revised Directive should contain, with reference to a number of crucial issues. Such indecisiveness could lead to wide discrepancies in the approaches of the EC, the European Parliament and the EU Member States concerning many issues of importance to animal welfare and the implementation of alternatives to animal experiments. These concerns range from logistical issues, such as requirements for named officers in authorised establishments, and the recording and publishing of statistics on animal use, to ethical and scientific problems, including the use of non-human primates, local ethical review, and education and training on the essential link between the Three Rs concept and best scientific practice. In each case, the basis for our concerns is explained, and suggestions for improvements to be incorporated into the revised Directive are made, in the hope that the harmonisation of approaches to laboratory animal experimentation and the use of alternative methods in the Member States can be maximised.     

Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.