Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

Reversible Control by Vitamin D of Granulocytes and Bacteria in the Lungs of Mice: An Ovalbumin-Induced Model of Allergic Airway Disease

Vitamin D may be essential for restricting the development and severity of allergic diseases and asthma, but a direct causal link between vitamin D deficiency and asthma has yet to be established. We have developed a ‘low dose’ model of allergic airway disease induced by intraperitoneal injection with ovalbumin (1 µg) and aluminium hydroxide (0.2 mg) in which characteristics of atopic asthma are recapitulated, including airway hyperresponsiveness, antigen-specific immunoglobulin type-E and lung inflammation. We assessed the effects of vitamin D deficiency throughout life (from conception until adulthood) on the severity of ovalbumin-induced allergic airway disease in vitamin D-replete and -deficient BALB/c mice using this model. Vitamin D had protective effects such that deficiency significantly enhanced eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male but not female mice. Vitamin D also suppressed the proliferation and T helper cell type-2 cytokine-secreting capacity of airway-draining lymph node cells from both male and female mice. Supplementation of initially vitamin D-deficient mice with vitamin D for four weeks returned serum 25-hydroxyvitamin D to levels observed in initially vitamin D-replete mice, and also suppressed eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male mice. Using generic 16 S rRNA primers, increased bacterial levels were detected in the lungs of initially vitamin D-deficient male mice, which were also reduced by vitamin D supplementation. These results indicate that vitamin D controls granulocyte levels in the bronchoalveolar lavage fluid in an allergen-sensitive manner, and may contribute towards the severity of asthma in a gender-specific fashion through regulation of respiratory bacteria.     

Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation

The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Here, we show that Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways.     

Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.     

Calpain system regulates muscle mass and glucose transporter GLUT4 turnover

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.     

BGX: a fully Bayesian integrated approach to the analysis of Affymetrix GeneChip data

We present Bayesian hierarchical models for the analysis of Affymetrix GeneChip data. The approach we take differs from other available approaches in two fundamental aspects. Firstly, we aim to integrate all processing steps of the raw data in a common statistically coherent framework, allowing all components and thus associated errors to be considered simultaneously. Secondly, inference is based on the full posterior distribution of gene expression indices and derived quantities, such as fold changes or ranks, rather than on single point estimates. Measures of uncertainty on these quantities are thus available. The models presented represent the first building block for integrated Bayesian Analysis of Affymetrix GeneChip data: the models take into account additive as well as multiplicative error, gene expression levels are estimated using perfect match and a fraction of mismatch probes and are modeled on the log scale. Background correction is incorporated by modeling true signal and cross-hybridization explicitly, and a need for further normalization is considerably reduced by allowing for array-specific distributions of nonspecific hybridization. When replicate arrays are available for a condition, posterior distributions of condition-specific gene expression indices are estimated directly, by a simultaneous consideration of replicate probe sets, avoiding averaging over estimates obtained from individual replicate arrays. The performance of the Bayesian model is compared to that of standard available point estimate methods on subsets of the well known GeneLogic and Affymetrix spike-in data. The Bayesian model is found to perform well and the integrated procedure presented appears to hold considerable promise for further development.     

Stereoscopic Vision in the Absence of the Lateral Occipital Cortex

Both dorsal and ventral cortical visual streams contain neurons sensitive to binocular disparities, but the two streams may underlie different aspects of stereoscopic vision. Here we investigate stereopsis in the neurological patient D.F., whose ventral stream, specifically lateral occipital cortex, has been damaged bilaterally, causing profound visual form agnosia. Despite her severe damage to cortical visual areas, we report that DF''s stereo vision is strikingly unimpaired. She is better than many control observers at using binocular disparity to judge whether an isolated object appears near or far, and to resolve ambiguous structure-from-motion. DF is, however, poor at using relative disparity between features at different locations across the visual field. This may stem from a difficulty in identifying the surface boundaries where relative disparity is available. We suggest that the ventral processing stream may play a critical role in enabling healthy observers to extract fine depth information from relative disparities within one surface or between surfaces located in different parts of the visual field.     

Automatic annotation of protein motif function with Gene Ontology terms

Background  

Conserved protein sequence motifs are short stretches of amino acid sequence patterns that potentially encode the function of proteins. Several sequence pattern searching algorithms and programs exist foridentifying candidate protein motifs at the whole genome level. However, amuch needed and importanttask is to determine the functions of the newly identified protein motifs. The Gene Ontology (GO) project is an endeavor to annotate the function of genes or protein sequences with terms from a dynamic, controlled vocabulary and these annotations serve well as a knowledge base.