A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening

Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.     

Evolutionary relationships among cyst-forming coccidia Sarcocystis spp. (Alveolata: Apicomplexa: Coccidea) in endemic African tree vipers and perspective for evolution of heteroxenous life cycle

Cyst-forming coccidia of the genus Sarcocystis (Alveolata: Apicomplexa: Coccidea) parasitize vertebrates worldwide. Data from the small subunit rRNA genes (SSU) and the D2 domain of the large subunit rRNA genes were used to reconstruct phylogeny for all species in the Sarcocystidae for which sequences are currently available. We have focused on the evolutionary history of species that circulate between snakes as definitive hosts and rodents as intermediate hosts. Trees were reconstructed using maximum parsimony, minimum evolution, maximum likelihood and the bayesian phylogenetics. Our reconstructions support monophyly of Sarcocystidae but fail to robustly resolve the relationship within clades. Using a concatenated dataset of available rDNAs, the "isosporoid" coccidia Neospora, Toxoplasma, Besnoitia, Isospora and Hyaloklossia form a sister group to the monophyletic Sarcocystis. Moreover, we show that Sarcocystis from arboreal vipers of the genus Atheris, which are endemic to the mountain rain forests of the Equatorial Africa, are monophyletic, with sister species parasitizing the desert viper Pseudocerastes persicus from the Near East. We report the co-evolution of Sarcocystis spp. with their final snake hosts. The geological history of the African continent, mountain ranges, forests and general SSU rDNA rates were used to construct a linearized tree. Possible origin of the heteroxenous life cycle of Sarcocystis is discussed.     

The effect of genomic information on optimal contribution selection in livestock breeding programs

Background

Long-term benefits in animal breeding programs require that increases in genetic merit be balanced with the need to maintain diversity (lost due to inbreeding). This can be achieved by using optimal contribution selection. The availability of high-density DNA marker information enables the incorporation of genomic data into optimal contribution selection but this raises the question about how this information affects the balance between genetic merit and diversity.

Methods

The effect of using genomic information in optimal contribution selection was examined based on simulated and real data on dairy bulls. We compared the genetic merit of selected animals at various levels of co-ancestry restrictions when using estimated breeding values based on parent average, genomic or progeny test information. Furthermore, we estimated the proportion of variation in estimated breeding values that is due to within-family differences.

Results

Optimal selection on genomic estimated breeding values increased genetic gain. Genetic merit was further increased using genomic rather than pedigree-based measures of co-ancestry under an inbreeding restriction policy. Using genomic instead of pedigree relationships to restrict inbreeding had a significant effect only when the population consisted of many large full-sib families; with a half-sib family structure, no difference was observed. In real data from dairy bulls, optimal contribution selection based on genomic estimated breeding values allowed for additional improvements in genetic merit at low to moderate inbreeding levels. Genomic estimated breeding values were more accurate and showed more within-family variation than parent average breeding values; for genomic estimated breeding values, 30 to 40% of the variation was due to within-family differences. Finally, there was no difference between constraining inbreeding via pedigree or genomic relationships in the real data.

Conclusions

The use of genomic estimated breeding values increased genetic gain in optimal contribution selection. Genomic estimated breeding values were more accurate and showed more within-family variation, which led to higher genetic gains for the same restriction on inbreeding. Using genomic relationships to restrict inbreeding provided no additional gain, except in the case of very large full-sib families.     

Using the realized relationship matrix to disentangle confounding factors for the estimation of genetic variance components of complex traits

Background

In the analysis of complex traits, genetic effects can be confounded with non-genetic effects, especially when using full-sib families. Dominance and epistatic effects are typically confounded with additive genetic and non-genetic effects. This confounding may cause the estimated genetic variance components to be inaccurate and biased.

Methods

In this study, we constructed genetic covariance structures from whole-genome marker data, and thus used realized relationship matrices to estimate variance components in a heterogenous population of ~ 2200 mice for which four complex traits were investigated. These mice were genotyped for more than 10,000 single nucleotide polymorphisms (SNP) and the variances due to family, cage and genetic effects were estimated by models based on pedigree information only, aggregate SNP information, and model selection for specific SNP effects.

Results and conclusions

We show that the use of genome-wide SNP information can disentangle confounding factors to estimate genetic variances by separating genetic and non-genetic effects. The estimated variance components using realized relationship were more accurate and less biased, compared to those based on pedigree information only. Models that allow the selection of individual SNP in addition to fitting a relationship matrix are more efficient for traits with a significant dominance variance.     

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.     

Regulation of Wnt signaling by the tumor suppressor adenomatous polyposis coli does not require the ability to enter the nucleus or a particular cytoplasmic localization

Wnt signaling plays key roles in development and disease. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling. Its best-characterized role is as part of the destruction complex, targeting the Wnt effector β-catenin (βcat) for phosphorylation and ultimate destruction, but several studies suggested APC also may act in the nucleus at promoters of Wnt-responsive genes or to shuttle βcat out for destruction. Even in its role in the destruction complex, APC's mechanism of action remains mysterious. We have suggested APC positions the destruction complex at the appropriate subcellular location, facilitating βcat destruction. In this study, we directly tested APC's proposed roles in the nucleus or in precisely localizing the destruction complex by generating a series of APC2 variants to which we added tags relocalizing otherwise wild-type APC to different cytoplasmic locations. We tested these for function in human colon cancer cells and Drosophila embryos. Strikingly, all rescue Wnt regulation and down-regulate Wnt target genes in colon cancer cells, and most restore Wnt regulation in Drosophila embryos null for both fly APCs. These data suggest that APC2 does not have to shuttle into the nucleus or localize to a particular subcellular location to regulate Wnt signaling.     

Some aspects of the biology of the stargazer mountain catfish,Amphilius uranoscopus (pfeffer); (Siluriformes: Amphiliidae) indigenous to Kenya streams

A study on some biological parameters of the mountain catfish, Amphilius uranoscopus Pfeffer 1889 (Silurifomes: Amphiliidae), was carried out in the Thego stream on the slopes of Mount Kenya from February to December 2002. Physical and chemical profiles of the Thego show that the water quality parameters is typical of high altitude streams with temperatures rarely exceeding 18°C, DO ranging from 7.9 to 8.2 mg l^?1 and relatively high conductivity (97–137 μS cm^?1) typical of perturbed lotic environments. A total of 1010 fish were caught by an electro‐fisher, with sizes ranging between 8 and 24 cm fork length. The population structure had a unimodal distribution with maxima at 14–16 cm. The length–weight relationship showed relatively narrow range in the slope ranging from 2.61 in April to 2.98 in February 2002, thereby suggesting isometric growth pattern. The fitted growth pattern of A. uranoscopus showed an asymptotic length (L_∞) of 28.5 cm and a growth curvature (K) of 0.56 year^?1 resulting in an estimated natural mortality coefficient (M) of 0.90 year^?1. The Fulton’s condition factor (K) was also relatively stable with a peak in April (0.92 ± 0.21) and lowest value in June (0.86 ± 0.10). As A. uranoscopus is not under commercial exploitation, the seemingly depressed population is possibly attributed to the introduced exotic rainbow trout that heavily predates on the species and environmental perturbations arising from changes in land use. The implications of such changes on A. uranoscopus are discussed.     

Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL

PINK1 is a mitochondrial kinase mutated in some familial cases of Parkinson's disease. It has been found to work in the same pathway as the E3 ligase Parkin in the maintenance of flight muscles and dopaminergic neurons in Drosophila melanogaster and to recruit cytosolic Parkin to mitochondria to mediate mitophagy in mammalian cells. Although PINK1 has a predicted mitochondrial import sequence, its cellular and submitochondrial localization remains unclear in part because it is rapidly degraded. In this study, we report that the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (PARL) mediates cleavage of PINK1 dependent on mitochondrial membrane potential. In the absence of PARL, the constitutive degradation of PINK1 is inhibited, stabilizing a 60-kD form inside mitochondria. When mitochondrial membrane potential is dissipated, PINK1 accumulates as a 63-kD full-length form on the outer mitochondrial membrane, where it can recruit Parkin to impaired mitochondria. Thus, differential localization to the inner and outer mitochondrial membranes appears to regulate PINK1 stability and function.     

Genome wide QTL mapping to identify candidate genes for carcass traits in Hanwoo (Korean Cattle)

Meat quality traits are the most economically important traits affecting the beef industry in Korea. We performed a whole genome quantitative trait locus (QTL) mapping study of carcass data in Hanwoo Korean cattle. Two hundred sixty-six Hanwoo steers from 65 sires were genotyped using a 10K Affymetrix SNP chip. The average SNP interval across the bovine genome was 1.5Mb. Associations between each individual SNP and four carcass traits [carcass weight (CWT), eye muscle area (EMA), back fat thickness (BFT), and marbling (MAR)] were assessed using a linear mixed model of each trait. Combined linkage and linkage disequilibrium analysis (LDLA) detected six potential QTL on BTA04, 06, 13, 16, 17, and 23 at the chromosome-wise level (P<0.05). Two MAR QTL were detected at 52.2 cM of BTA06 and 46.04 cM of BTA17. We identified three genes (ARAP2, LOC539460, and LOC511424) in the QTL region of BTA06 and seven genes (RPS14, SCARB1, LOC782103, BRI3BP, AACS, DHX37, and UBC) in the QTL region of BTA17. One significant QTL for CWT was detected at 100 cM on BTA04 and the corresponding QTL region spanned 1.7 cM from 99.7 to 101.4 cM. For EMA QTL, one significant QTL was detected at 3.9 cM of BTA23 and the most likely QTL interval was 1.4 cM, placing 15 candidate genes in the marker bracket. Finally, two QTL for BFT were identified at 68 cM on BTA13 and 24 cM on BTA16. The LPIN3 gene, which is functionally associated with lipodystrophy in humans, is located in the BFT QTL on BTA13. Thus, two potential candidate genes, acetoacetyl-CoA synthetase (AACS) and lipin (LPIN), were detected in QTL regions on BTA17 for MAR and BTA13 for BFT, respectively. In conclusion, LDLA analysis can be used to detect chromosome regions harboring QTL and candidate genes with a low density SNP panel, yielding relatively narrow confidence intervals regarding location.