Inactive forms of the catalytic subunit of protein kinase A are expressed in the brain of higher primates

It is well documented that the beta-gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N-terminal end encoded by alternative use of several exons located 5' to exon 2 in the human, bovine and mouse Cbeta gene. In the present study, we demonstrate the expression of six novel human Cbeta mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbetaDelta4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbetaDelta4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbetaDelta4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP-responsive element binding protein. Furthermore, co-expression of CbetaDelta4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CbetaDelta4 variants associate with both RI and RII in a cAMP-independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CbetaDelta4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.     

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.     

Identification of the yellow skin gene reveals a hybrid origin of the domestic chicken

Yellow skin is an abundant phenotype among domestic chickens and is caused by a recessive allele (W*Y) that allows deposition of yellow carotenoids in the skin. Here we show that yellow skin is caused by one or more cis-acting and tissue-specific regulatory mutation(s) that inhibit expression of BCDO2 (beta-carotene dioxygenase 2) in skin. Our data imply that carotenoids are taken up from the circulation in both genotypes but are degraded by BCDO2 in skin from animals carrying the white skin allele (W*W). Surprisingly, our results demonstrate that yellow skin does not originate from the red junglefowl (Gallus gallus), the presumed sole wild ancestor of the domestic chicken, but most likely from the closely related grey junglefowl (Gallus sonneratii). This is the first conclusive evidence for a hybrid origin of the domestic chicken, and it has important implications for our views of the domestication process.     

Phylogeny and taxonomy of the genus Phylloporia (Hymenochaetales)

Based on 28 taxa, including six species of Phylloporia, and respectively one representative of 17 genera of the Hymenochaetales, a phylogenetic analysis of a region of the large subunit of the nuclear encoded ribosomal DNA was performed. Molecular sequence data as well as morphological and anatomical features show the genus to be monophyletic. Next related to Phylloporia is Fulvifomes. The phylogenetic relationships of Phylloporia are discussed. In addition the genus Phylloporia is monographed; 12 species are accepted and described with a key.     

The use of Endo-Porter to deliver morpholinos in kidney organ culture

Cellular interactions in development of the kidney are used as a model of reciprocal inductive events between epithelium and mesenchyme. Time- and labor-intensive methods have been developed to study this phenomenon. For example, in mice, the targeted disruption of genes in vivo has been used to modify the genetic program directing kidney development. However, gene targeting is a resource-intensive approach and alternative strategies for gene and protein modification in the kidney need to be developed. Herein, we have developed an efficient system for the delivery of antisense morpholino to alter normal protein expression. We describe the use of Endo-Porter to effectively deliver morpholinos to all parts and regions of the kidney explant. Also, we definitively show via confocal microscopy and Western blot analysis that the use of Endo-Porter in delivering antisense morpholinos is robust throughout the entire kidney explant, providing efficient suppression of protein expression. This method saves time and cost when compared with targeted disruption and is an improvement upon previous kidney organ culture methods.     

Metal ion accessibility of histidine-modified superfolder green fluorescent protein expressed in Escherichia coli

Green fluorescent protein (GFP) is frequently utilized for metal ion detection and quantification. To improve the metal binding potential of GFP, three residues (N146, F165, and L201) were substituted to histidines. Each variant responded differently upon interaction with metal ions. More than 80% of N146H, having the most accessible surface area, could bind to immobilized metal ions. However, only F165H exhibited significant differences in quenching by soluble metal ions (22% fluorescence decrease) in comparison with the template protein (12%). These findings can be utilized for designing GFP variants for metal binding and sensor applications.     

Bayesian calibration method used to elucidate carbon turnover in forest on drained organic soil

Depending on the balance between sink and source processes for C, drained organic forest soil ecosystems can be in balance or act as net sinks or sources of CO₂ to the atmosphere. In order to study the effect of groundwater level and soil temperature on C-flux, the CoupModel was calibrated (climate data, groundwater levels, soil CO₂ flux, net ecosystem fluxes of CO₂-exchange, sensible heat flux and latent heat flux, forest production etc.) for a drained forest in Sweden. Bayesian calibration techniques were used to elucidate how different parameters and variables were interlinked in C-circulation. The calibrated model reproduced abiotic and biotic variables reasonably well except for root respiration, which was largely underestimated. Bayesian calibration reduced the uncertainties in the model and highlighted the fact that calibrations should be performed with a high number of parameters instead of specific parameter values.     

Long-term changes in floristic diversity in southern Sweden: palynological richness, vegetation dynamics and land-use

The rarefaction technique is applied to two Holocene pollen sequences (covering the last 12,000 calendar years) from two lakes in southern Sweden. One represents an open agricultural landscape, the other a partly wooded and less cultivated landscape. The inferred palynological richness is interpreted as an approximate measure of floristic diversity at the landscape scale. The overall trend is an increased diversity from the mid-Holocene to the Modern period, which is linked to a parallel rise in human impact. The pattern is similar for the two sites with peaks corresponding to archaeological periods characterised by deforestation and expanding settlement and agriculture. The highest diversity was reached during the Medieval period, about a.d. 1,000–1,400. Declining diversity during the last 200 years characterises the agrarian landscape. These results confirm, for southern Scandinavia, the “intermediate disturbance” hypothesis for biodiversity at the landscape scale and on millennial to century time scales. They have implications for landscape management in modern nature conservation that has the purpose of maintaining and promoting biodiversity.     

3D-Structure and function of strictosidine synthase--the key enzyme of monoterpenoid indole alkaloid biosynthesis

Strictosidine synthase (STR; EC 4.3.3.2) plays a key role in the biosynthesis of monoterpenoid indole alkaloids by catalyzing the Pictet-Spengler reaction between tryptamine and secologanin, leading exclusively to 3alpha-(S)-strictosidine. The structure of the native enzyme from the Indian medicinal plant Rauvolfia serpentina represents the first example of a six-bladed four-stranded beta-propeller fold from the plant kingdom. Moreover, the architecture of the enzyme-substrate and enzyme-product complexes reveals deep insight into the active centre and mechanism of the synthase highlighting the importance of Glu309 as the catalytic residue. The present review describes the 3D-structure and function of R. serpentina strictosidine synthase and provides a summary of the strictosidine synthase substrate specificity studies carried out in different organisms to date. Based on the enzyme-product complex, this paper goes on to describe a rational, structure-based redesign of the enzyme, which offers the opportunity to produce novel strictosidine derivatives which can be used to generate alkaloid libraries of the N-analogues heteroyohimbine type. Finally, alignment studies of functionally expressed strictosidine synthases are presented and the evolutionary aspects of sequence- and structure-related beta-propeller folds are discussed.