In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.     

The origin and evolution of stereotyped patterns of macrochaetes on the nota of cyclorraphous Diptera

A long-standing problem in evolutionary biology is how genetic variation arises within populations and evolves to make species anatomically different. Many of the morphological differences in body plans between animal groups are thought to result from changes in gene expression during development. The rules governing the structure and evolution of cis-regulatory gene sequences are unknown, however, and the evolution of traits between closely related species remains relatively unexplored at a molecular level. To study the evolution of gene regulation, it is necessary to find a tractable trait that varies between species and for which the genetic regulation is well known in at least one of the species. The stereotyped, two-dimensional pattern of bristles on the thorax of Drosophila has been intensively investigated and is due to a precise spatial expression of proneural genes. Other species of flies have different bristle patterns and so comparisons between them provide a good paradigm for the study of changes in gene regulation. Here, we review the current state of understanding of these changes.     

Cellular Localisation of Adenylyl Cyclase: A Post-genome Perspective

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.Invocation: This paper was written to honour one of the founders of chemical neuroanatomy—Professor Miklós Palkovits on his 70th birthday.     

Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of "primitive" eukaryotes

Many of the protists thought to represent the deepest branches on the eukaryotic tree are assigned to a loose assemblage called the "excavates." This includes the mitochondrion-lacking diplomonads and parabasalids (e.g., Giardia and Trichomonas) and the jakobids (e.g., Reclinomonas). We report the first multigene phylogenetic analyses to include a comprehensive sampling of excavate groups (six nuclear-encoded protein-coding genes, nine of the 10 recognized excavate groups). Excavates coalesce into three clades with relatively strong maximum likelihood bootstrap support. Only the phylogenetic position of Malawimonas is uncertain. Diplomonads, parabasalids, and the free-living amitochondriate protist Carpediemonas are closely related to each other. Two other amitochondriate excavates, oxymonads and Trimastix, form the second monophyletic group. The third group is comprised of Euglenozoa (e.g., trypanosomes), Heterolobosea, and jakobids. Unexpectedly, jakobids appear to be specifically related to Heterolobosea. This tree topology calls into question the concept of Discicristata as a supergroup of eukaryotes united by discoidal mitochondrial cristae and makes it implausible that jakobids represent an independent early-diverging eukaryotic lineage. The close jakobids-Heterolobosea-Euglenozoa connection demands complex evolutionary scenarios to explain the transition between the presumed ancestral bacterial-type mitochondrial RNA polymerase found in jakobids and the phage-type protein in other eukaryotic lineages, including Euglenozoa and Heterolobosea.     

Discontinuity induced bifurcations in a model of Saccharomyces cerevisiae

We perform a bifurcation analysis of the mathematical model of Jones and Kompala [K.D. Jones, D.S. Kompala, Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures, J. Biotechnol. 71 (1999) 105-131]. Stable oscillations arise via Andronov-Hopf bifurcations and exist for intermediate values of the dilution rate as has been noted from experiments previously. A variety of discontinuity induced bifurcations arise from a lack of global differentiability. We identify and classify discontinuous bifurcations including several codimension-two scenarios. Bifurcation diagrams are explained by a general unfolding of these singularities.     

Three hundred and fifty generations of extreme food specialisation: testing predictions of nutritional ecology

We used a strain of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), that had been reared for approximately 350 generations in a precisely characterised environment to test hypotheses regarding the influence of nutritional heterogeneity on the evolution of nutrient regulatory responses. Caterpillars were maintained with ad libitum access to a diet that emulated that of an extreme nutritional specialist, comprising a homogeneous food of fixed nutrient composition. We measured performance (survival, development rate, and pupal mass), as well as the protein and carbohydrate intake of individual caterpillars confined to one of a range of single foods differing in their protein, carbohydrate, and water content. In a separate experiment, we measured the amount and balance of protein and carbohydrate self-selected by caterpillars presented with nutritionally complementary foods. Results showed a close fit with three of four predictions about the nutritional responses of 'nutrient specialist' feeders: (1) survival, development rate, and pupal mass were highest for animals given diets with the protein:carbohydrate composition of the ancestral culture diet, and dropped off sharply with higher and lower protein:carbohydrate balance, (2) caterpillars coped poorly with dietary dilution by water, irrespective of the macronutrient balance, and (3) the self-selected intake point corresponded with the macronutrient balance that gave peak performance (i.e., that of the ancestral culture diet). The fourth prediction, that caterpillars would be disinclined to over-ingest nutrients on imbalanced diets, was at best weakly met. We hypothesise that the evolution and maintenance of the specialist strategy might, paradoxically, require some degree of environmental heterogeneity.     

The Folding Pathway of the Antibody VL Domain

Antibodies are modular proteins consisting of domains that exhibit a β-sandwich structure, the so-called immunoglobulin fold. Despite structural similarity, differences in folding and stability exist between different domains. In particular, the variable domain of the light chain V_L is unusual as it is associated with misfolding diseases, including the pathologic assembly of the protein into fibrillar structures. Here, we have analysed the folding pathway of a V_L domain with a view to determine features that may influence the relationship between productive folding and fibril formation. The V_L domain from MAK33 (murine monoclonal antibody of the subtype κ/IgG1) has not previously been associated with fibrillisation but is shown here to be capable of forming fibrils. The folding pathway of this V_L domain is complex, involving two intermediates in different pathways. An obligatory early molten globule-like intermediate with secondary structure but only loose tertiary interactions is inferred. The native state can then be formed directly from this intermediate in a phase that can be accelerated by the addition of prolyl isomerases. However, an alternative pathway involving a second, more native-like intermediate is also significantly populated. Thus, the protein can reach the native state via two distinct folding pathways. Comparisons to the folding pathways of other antibody domains reveal similarities in the folding pathways; however, in detail, the folding of the V_L domain is striking, with two intermediates populated on different branches of the folding pathway, one of which could provide an entry point for molecules diverted into the amyloid pathway.     

Spatial distribution of the active surveillance of sheep scrapie in Great Britain: an exploratory analysis

Background  

This paper explores the spatial distribution of sampling within the active surveillance of sheep scrapie in Great Britain. We investigated the geographic distribution of the birth holdings of sheep sampled for scrapie during 2002 – 2005, including samples taken in abattoir surveys (c. 83,100) and from sheep that died in the field ("fallen stock", c. 14,600). We mapped the birth holdings by county and calculated the sampling rate, defined as the proportion of the holdings in each county sampled by the surveys. The Moran index was used to estimate the global spatial autocorrelation across Great Britain. The contributions of each county to the global Moran index were analysed by a local indicator of spatial autocorrelation (LISA).     

Birth weight,sex and childhood cancer: A report from the United Kingdom Childhood Cancer Study

Birth weight has been linked to the risk of developing childhood cancer, in particular childhood leukaemia. However, despite many childhood cancers having a male predominance and boys generally weighing more than girls at birth few studies have reported sex-specific associations. The relationship between birth weight and childhood cancer risk was examined using information from a national case-control study. Children (0–14 years) newly diagnosed with cancer in GB were ascertained between 1991 and 1996 (n = 3651) and for comparison, controls matched on sex, month and year of birth were identified from primary care population registers (n = 6337). Birth weights were obtained from the Office of National Statistics for all targeted subjects born in England and Wales. Overall, cases were, on average, 30 g heavier at birth than controls (p = 0.003) with differences seen by cancer type; those diagnosed with hepatic tumours weighing around 500 g less than controls at birth (p < 0.0001) and those with leukaemia being, on average, 50 g heavier than those without (p = 0.001). An interaction between birth weight and sex was found for acute leukaemia (χ² = 11.2, p = 0.04) and when data were stratified by sex, an association between high birth weight and risk of ALL was seen with girls (>4000 g, OR 1.86, 95% CI 1.38–2.50, χ² for trend 20.2, p < 0.0001). Our results support the hypothesis that birth weight is an important determinant for childhood cancer. In addition, the data are consistent with the notion that childhood leukaemia has a prenatal origin.