The regulation of protein synthesis in animal cells

The technique of DNA-RNA molecular hybridization was adopted for investigating the regulation of protein synthesis at the genome level in differentiated animal cells. In preliminary experiments, attempts were made to assay mammalian DNA-RNA complexes by means of methylated albumin-kieselguhr chromatography, Sephadex G-200 chromatography and by selective adsorption on nitrocellulose filters. The latter technique proved to be the most convenient for the present studies, especially when the DNA was immobilised to the nitrocellulose filter prior to hybridisation with RNA. Using this technique for the hybridization of in vivo labelled 14C- rat kidney RNA to homologous DNA it was found that under conditions of maximum hybridization only 7% of the DNA formed hybrids with the RNA. This result was taken as evidence for the restriction of DNA template activity in animal cells. In an attempt to detect tissue specific RNAs in various rat organs, comparisons were made of the rapidly 32P-labelled RNA factions of rat liver, kidney and pancreas by competitive hybridisation against unlabelled rat kidney RNA. Differneces were observed in the hybridization kinetics of the RNAs from each organ. As it was not possible to determine the real specific activity of rapidly labelled RNA from in vivo labelling experiments, it was concluded that the observed differences in competitive hybridization kinetics of these RNAs could be due either to absolute differences in RNA types or due to differences in the specific activities of the RNAs isolated from each organ. The difficulties associated with the use of in vivo labelled RNAs could be avoided by synthesising the RNAs in vitro from chromatin primers with the RNA polymerase of Micrococcus lysodeikticus. Experiments in which the RNA synthesised in vitro from calf thymus and rabbit thymus and bone marrow chromatins was hybridized to homologous DNA, showed that the template activity of the isolated chromatin was restricted to 5-10% of the total DNA. Competitive hybridization experiments showed that the RNA synthesised in vivo from calf thymus chromatin was qualitatively identical to that synthesised in the in vitro system. This was also confirmed with the RNAs synthesised in vivo and in vitro from rabbit bone marrow chromatin. It was concluded that the restriction of DNA template activity found in vivo in animal cells is also a property of the isolated chromatin. In further competitive hybridization experiments, the RNAs synthesised in vivo from rabbit thymus and bone marrow were compared separately with the RNAs synthesised in vitro from rabbit thymus and bone marrow chromatins. Evidence was provided for the existence of qualitative differences in the RNA populations of the two tissues. This suggests that the restriction of DNA template activity in differentiated cells is organ specific. Investigations into the nature of the specific restriction of template activity in calf thymus chromatin suggested that protein ionically bound to DNA is responsible for the restriction. Under specified conditions it was found possible to reconstitute chromatin from crude DNA, histone and non-histone fractions. The uncleohistone obtained by reconstituting DNA with the histone fraction alone, could not direct the in vitro synthesis of any RNA capable of hybridising to calf thymus DNA. On the other hand, nucleohistone reconstituted from DNA in the presence of both histone and non-histone fractions resulted in a specific part of the DNA being available for transcription. The magnitude of this unrestricted portion was found to be the same as that of whole chromatin. These results suggest that there exists in calf thymus chromatin a non-histone fraction responsible for conferring the specific restriction of DNA template activity

Estimating variance components and predicting breeding values for eventing disciplines and grades in sport horses

Eventing competitions in Great Britain (GB) comprise three disciplines, each split into four grades, yielding 12 discipline-grade traits. As there is a demand for tools to estimate (co)variance matrices with a large number of traits, the aim of this work was to investigate different methods to produce large (co)variance matrices using GB eventing data. Data from 1999 to 2008 were used and penalty points were converted to normal scores. A sire model was utilised to estimate fixed effects of gender, age and class, and random effects of sire, horse and rider. Three methods were used to estimate (co)variance matrices. Method 1 used a method based on Gibbs sampling and data augmentation and imputation. Methods 2a and 2b combined sub-matrices from bivariate analyses; one took samples from a multivariate Normal distribution defined by the covariance matrix from each bivariate analysis, then analysed these data in a 12-trait multivariate analysis; the other replaced negative eigenvalues in the matrix with positive values to obtain a positive definite (co)variance matrix. A formal comparison of models could not be conducted; however, estimates from all methods, particularly Methods 2a/2b, were in reasonable agreement. The computational requirements of Method 1 were much less compared with Methods 2a or 2b. Method 2a heritability estimates were as follows: for dressage 7.2% to 9.0%, for show jumping 8.9% to 16.2% and for cross-country 1.3% to 1.4%. Method 1 heritability estimates were higher for the advanced grades, particularly for dressage (17.1%) and show jumping (22.6%). Irrespective of the model, genetic correlations between grades, for dressage and show jumping, were positive, high and significant, ranging from 0.59 to 0.99 for Method 2a and 0.78 to 0.95 for Method 1. For cross-country, using Method 2a, genetic correlations were only significant between novice and pre-novice (0.75); however, using Method 1 estimates were all significant and low to moderate (0.36 to 0.70). Between-discipline correlations were all low and of mixed sign. All methods produced positive definite 12312 (co)variance matrices, suitable for the prediction of breeding values. Method 1 benefits from much reduced computational requirements, and by performing a true multivariate analysis

Thrifty metabolic programming in rats is induced by both maternal undernutrition and postnatal leptin treatment, but masked in the presence of both: implications for models of developmental programming.

BACKGROUND: Maternal undernutrition leads to an increased risk of metabolic disorders in offspring including obesity and insulin resistance, thought to be due to a programmed thrifty phenotype which is inappropriate for a subsequent richer nutritional environment. In a rat model, both male and female offspring of undernourished mothers are programmed to become obese, however postnatal leptin treatment gives discordant results between males and females. Leptin treatment is able to rescue the adverse programming effects in the female offspring of undernourished mothers, but not in their male offspring. Additionally, in these rats, postnatal leptin treatment of offspring from normally-nourished mothers programmes their male offspring to develop obesity in later life, while there is no comparable effect in their female offspring. RESULTS: We show by microarray analysis of the female liver transcriptome that both maternal undernutrition and postnatal leptin treatment independently induce a similar thrifty transcriptional programme affecting carbohydrate metabolism, amino acid metabolism and oxidative stress genes. Paradoxically, however, the combination of both stimuli restores a more normal transcriptional environment. This demonstrates that "leptin reversal" is a global phenomenon affecting all genes involved in fetal programming by maternal undernourishment and leptin treatment. The thrifty transcriptional programme was associated with pro-inflammatory markers and downregulation of adaptive immune mediators, particularly MHC class I genes, suggesting a deficit in antigen presentation in these offspring. CONCLUSIONS: We propose a revised model of developmental programming reconciling the male and female observations, in which there are two competing programmes which collectively drive liver transcription. The first element is a thrifty metabolic phenotype induced by early life growth restriction independently of leptin levels. The second is a homeostatic set point calibrated in response to postnatal leptin surge, which is able to over-ride the metabolic programme. This "calibration model" for the postnatal leptin surge, if applicable in humans, may have implications for understanding responses to catch-up growth in infants. Additionally, the identification of an antigen presentation deficit associated with metabolic thriftiness may relate to a previously observed correlation between birth season (a proxy for gestational undernutrition) and infectious disease mortality in rural African communities

Neonatal pigs are susceptible to experimental Zika virus infection

Emerging Microbes & Infections (2017) 6, e6; doi:10.1038/emi.2016.133; published online 15 February 201

The transcriptional activator Gli2 modulates T-cell receptor signalling through attenuation of AP-1 and NFκB activity

Different tissues contain diverse and dynamic cellular niches, providing distinct signals to tissue-resident or migratory infiltrating immune cells. Hedgehog (Hh) proteins are secreted inter-cellular signalling molecules, which are essential during development and are important in cancer, post-natal tissue homeostasis and repair. Hh signalling mediated by the Hh-responsive transcription factor Gli2 also has multiple roles in T-lymphocyte development and differentiation.Here, we investigate the function of Gli2 in T-cell signalling and activation. Gene transcription driven by the Gli2 transcriptional activator isoform (Gli2A) attenuated T-cell activation and proliferation following T-cell receptor (TCR) stimulation. Expression of Gli2A in T-cells altered gene expression profiles, impaired the TCR-induced Ca2+ flux and nuclear expression of NFAT2, suppressed upregulation of molecules essential for activation, and attenuated signalling pathways upstream of the AP-1 and NFκB complexes, leading to reduced activation of these important transcription factors. Inhibition of physiological Hh-dependent transcription increased NFκB activity upon TCR ligation. These data are important for nderstanding the molecular mechanisms of immunomodulation, particularly in tissues where Hh proteins or other Gli-activating ligands such as TGFβ are upregulated, including during inflammation, tissue damage and repair, and in tumour microenvironments

Coercion and conciliation at the edge of empire: state-building and its limits in Waziristan, 1849-1914

This paper proposes an alternative history of the colonial frontier. Focussing on Waziristan, the paper challenges prevailing understandings of colonial frontier policy by demonstrating important and widely-overlooked continuities in colonial engagements with the people and territory of the frontier

A Switch in Hepatic Cortisol Metabolism across the Spectrum of Non Alcoholic Fatty Liver Disease

Context: Non alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. NAFLD represents a spectrum of liver disease ranging from reversible hepatic steatosis, to non alcoholic steato-hepatitis (NASH) and cirrhosis. The potential role of glucocorticoids (GC) in the pathogenesis of NAFLD is highlighted in patients with GC excess, Cushing's syndrome, who develop central adiposity, insulin resistance and in 20% of cases, NAFLD. Although in most cases of NAFLD, circulating cortisol levels are normal, hepatic cortisol availability is controlled by enzymes that regenerate cortisol (F) from inactive cortisone (E) (11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1), or inactivate cortisol through A-ring metabolism (5α- and 5β-reductase, 5αR and 5βR). Objective and Methods: In vitro studies defined 11β-HSD1 expression in normal and NASH liver samples. We then characterised hepatic cortisol metabolism in 16 patients with histologically proven NAFLD compared to 32 obese controls using gas chromatographic analysis of 24 hour urine collection and plasma cortisol generation profile following oral cortisone. Results: In patients with steatosis 5αR activity was increased, with a decrease in hepatic 11β-HSD1 activity. Total cortisol metabolites were increased in this group consistent with increased GC production rate. In contrast, in patients with NASH, 11β-HSD1 activity was increased both in comparison to patients with steatosis, and controls. Endorsing these findings, 11β-HSD1 mRNA and immunostaining was markedly increased in NASH patients in peri septal hepatocytes and within CD68 positive macrophages within inflamed cirrhotic septa. Conclusion: Patients with hepatic steatosis have increased clearance and decreased hepatic regeneration of cortisol and we propose that this may represent a protective mechanism to decrease local GC availability to preserve hepatic metabolic phenotype. With progression to NASH, increased 11β-HSD1 activity and consequent cortisol regeneration may serve to limit hepatic inflammation