Structural requirements for the BARD1 tumor suppressor in chromosomal stability and homology-directed DNA repair

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.     

Identification of unique molecular subdomains in the perichondrium and periosteum and their role in regulating gene expression in the underlying chondrocytes

Developing cartilaginous and ossified skeletal anlagen is encapsulated within a membranous sheath of flattened, elongated cells called, respectively, the perichondrium and the periosteum. These periskeletal tissues are organized in distinct morphological layers that have been proposed to support distinct functions. Classical experiments, particularly those using an in vitro organ culture system, demonstrated that these tissues play important roles in regulating the differentiation of the subjacent skeletal elements. However, there has been a lack of molecular markers that would allow analysis of these interactions. To understand the molecular bases for the roles played by the periskeletal tissues, we generated microarrays from perichondrium and periosteum cDNA libraries and used them to compare the gene expression profiles of these two tissues. In situ hybridization analysis of genes identified on the microarrays revealed many unique markers for these tissues and demonstrated that the histologically distinct layers of the perichondrium and periosteum are associated with distinct molecular expression domains. Moreover our marker analysis identified new domains that had not been previously recognized as distinct within these tissues as well as a previously uncharacterized molecular domain along the lateral edges of the adjacent developing cartilage that experimental analysis showed to be dependent upon the perichondrium.     

Perichondrial-mediated TGF-beta regulation of cartilage growth in avian long bone development

We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself.     

Evolution of the variable gene segments and recombination signal sequences of the human T-cell receptor α/δ locus

The T-cell receptor (TCR) and loci are particularly interesting because of their unique genomic structure, in that the gene segments for each locus are interspersed. The origin of this remarkable gene segment arrangement is obscure. In this report, we investigated the evolution of the TCR and variable loci and their respective recombination signal sequences (RSSs). Our phylogenetic analyses divided the and variable gene segments into two major groups each with distinguishing motifs in both the framework and complementarity determining regions (CDRs). Sequence analyses revealed that TCR variable segments share similar CDR2 sequences with immunoglobulin light chain variable segments, possibly revealing similar evolutionary histories. Maximum likelihood analysis of the region on Chromosome 14q11.2 containing the loci revealed two possible ancestral TCR / variable segments, TRDV2 and TRAV1-1/1-2, respectively. Maximum parsimony revealed different evolutionary patterns between the variable segment and RSS of the same variable gene arguing for dissimilar evolutionary origins. Two models could account for this difference: a V(D)J recombination activity involving embedded heptamer-like motifs in the germline genome, or, more plausibly, an unequal sister chromatid crossing-over. Either mechanism would have resulted in increased diversity for the adaptive immune system.     

Modernization and medicinal plant knowledge in a Caribbean horticultural village

Herbal medicine is the first response to illness in rural Dominica. Every adult knows several "bush" medicines, and knowledge varies from person to person. Anthropological convention suggests that modernization generally weakens traditional knowledge. We examine the effects of commercial occupation, consumerism, education, parenthood, age, and gender on the number of medicinal plants freelisted by individuals. All six predictors are associated with bush medical knowledge in bivariate analyses. Contrary to predictions, commercial occupation and consumerism are positively associated with herbal knowledge. Gender, age, occupation, and education are significant predictors in multivariate analysis. Women tend to recall more plants than do men. Education is negatively associated with plants listed; age positively associates with number of species listed. There are significant interactions among commercial occupation, education, age, and parenthood, suggesting that modernization has complex effects on knowledge of traditional medicine in Dominica.     

Contribution of hydrophobic interactions to protein stability

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mol per -CH₂- group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per -CH₂- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH₂- group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG_tr values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds contribute 40 ± 4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions.     

Negatively cooperative binding of high-density lipoprotein to the HDL receptor SR-BI

Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 μg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.     

Differences in candidate gene association between European ancestry and African American asthmatic children

Background

Candidate gene case-control studies have identified several single nucleotide polymorphisms (SNPs) that are associated with asthma susceptibility. Most of these studies have been restricted to evaluations of specific SNPs within a single gene and within populations from European ancestry. Recently, there is increasing interest in understanding racial differences in genetic risk associated with childhood asthma. Our aim was to compare association patterns of asthma candidate genes between children of European and African ancestry.

Methodology/Principal Findings

Using a custom-designed Illumina SNP array, we genotyped 1,485 children within the Greater Cincinnati Pediatric Clinic Repository and Cincinnati Genomic Control Cohort for 259 SNPs in 28 genes and evaluated their associations with asthma. We identified 14 SNPs located in 6 genes that were significantly associated (p-values <0.05) with childhood asthma in African Americans. Among Caucasians, 13 SNPs in 5 genes were associated with childhood asthma. Two SNPs in IL4 were associated with asthma in both races (p-values <0.05). Gene-gene interaction studies identified race specific sets of genes that best discriminate between asthmatic children and non-allergic controls.

Conclusions/Significance

We identified IL4 as having a role in asthma susceptibility in both African American and Caucasian children. However, while IL4 SNPs were associated with asthma in asthmatic children with European and African ancestry, the relative contributions of the most replicated asthma-associated SNPs varied by ancestry. These data provides valuable insights into the pathways that may predispose to asthma in individuals with European vs. African ancestry.     

The healthy heart race: a short-duration, hands-on activity in cardiovascular physiology for museums and science festivals

The "Healthy Heart Race" activity provides a hands-on demonstration of cardiovascular function suitable for lay audiences. It was field tested during the United States of America Science and Engineering Festival held in Washington, DC, in October 2010. The basic equipment for the activity consisted of lengths of plastic tubing, a hand pump, collection containers, clamps, and simulated blood prepared by tinting water with red food coloring. Student participants were first asked to experience the effort required to pump through an unaltered tube. A presenter then applied a strong clamp that pinched each tube downstream from the pump, and students were asked to pump against the increased resistance. The students' observations were then used as the basis for discussions of atherosclerosis and coronary heart disease with the presenters. Distribution of informative postcards during the 2 days of the festival indicated that at least 2,500 students completed the Healthy Heart Race activity. Our experiences to date suggest that the Healthy Heart Race activity can be accomplished effectively in the high-volume, high-distraction environment of a science fair or museum.