CTCF mediates insulator function at the CFTR locus

Regulatory elements that lie outside the basal promoter of a gene may be revealed by local changes in chromatin structure and histone modifications. The promoter of the CFTR (cystic fibrosis transmembrane conductance regulator) gene is not responsible for its complex pattern of expression. To identify important regulatory elements for CFTR we have previously mapped DHS (DNase I-hypersensitive sites) across 400 kb spanning the locus. Of particular interest were two DHS that flank the CFTR gene, upstream at -20.9 kb with respect to the translational start site, and downstream at +15.6 kb. In the present study we show that these two DHS possess enhancer-blocking activity and bind proteins that are characteristic of known insulator elements. The DHS core at -20.9 kb binds CTCF (CCCTC-binding factor) both in vitro and in vivo; however, the +15.6 kb core appears to bind other factors. Histone-modification analysis across the CFTR locus highlights structural differences between the -20.9 kb and +15.6 kb DHS, further suggesting that these two insulator elements may operate by distinct mechanisms. We propose that these two DHS mark the boundaries of the CFTR gene functional unit and establish a chromatin domain within which the complex profile of CFTR expression is maintained.     

A logical model provides insights into T cell receptor signaling

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.     

Identification of novel high-frequency DNA methylation changes in breast cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.     

Soil seed banks of two montane riparian areas: implications for restoration

Understanding the role of seed banks can be important for designing restoration projects. Using the seedling emergence method, we investigated the soil seed banks of two montane, deciduous riparian forest ecosystems of southeastern Arizona. We contrasted the seed banks and extant vegetation of Ramsey Canyon, which is the site of riparian restoration activities, with that of Garden Canyon, which has been less affected by human land uses. Fewer plant species were found at Ramsey Canyon than Garden Canyon, for both the seed bank and extant vegetation, and the vegetation at Ramsey Canyon (seed bank and extant) had consistently drier wetland indicator scores. As well, vegetation patterns within sampling zones (channel margins and adjacent riparian forests) differed between canyons. At Garden Canyon channel margins, the seed bank and extant vegetation had relatively high similarity, with herbaceous wetland perennial species dominating. Extant vegetation in the floodplain riparian forest zone at Garden Canyon had a drier wetland indicator score than the seed bank, suggesting that the floodplains are storing seeds dispersed from wetter fluvial surfaces. Vegetation patterns for Ramsey Canyon channel margins were similar to those for Garden Canyon floodplains. Vegetation patterns in the Ramsey Canyon riparian forest zone were indicative of non-flooded conditions with an abundance of upland species in the soil seed bank and extant vegetation. Channel geomorphology measurements indicated that much of the riparian forest zone at Ramsey Canyon is functionally a terrace, a condition that may be a legacy of channel erosion from historic land uses. Steep, erodible channel slopes may contribute to the low seed bank germinant density at Ramsey Canyon channel margins, and narrower flood-prone area may explain the greater terrestrialization of the vegetation in both sampling zones. We recommend testing the use of donor soils from more diverse stream reaches to restore biodiversity levels at Ramsey Canyon, following restoration activities such as channel-widening. Seed banks from Garden Canyon, for example, although predominantly consisting of herbaceous perennials, would supply species with a range of moisture tolerances, life spans, and growth forms. We also recommend that restorationists take care not to harm seed banks exposed during removal of introduced species; at Ramsey Canyon, soil seed banks were equally diverse in areas with high and low cover of the introduced Vinca major (a legacy of Ramsey Canyon land use).     

Normal hematological indices, blood chemistry and histology and ultrastructure of pancreatic islets in the wild Indian bonnet monkeys (Macaca radiata radiata)

The present study is aimed at determining some haematological and biochemical parameters in the wild Indian bonnet monkeys as also the microscopic and ultrastructural characteristics of their pancreatic islets. Adult wild Indian bonnet monkeys (Macaca radiata radiata) of both sexes weighing between 2.5 and 4 kg were used in these experiments. Their platelet, reticulocyte and total leukocyte counts and the blood concentrations of hemoglobin and plasma proteins and the serum concentrations of aspartate amino transferase, alanine amino transferase and calcium are similar to the values reported for M. mulatta. Plasma glucose is lower when compared with reported values of M. mulatta and M. fascicularis. Insulin levels are comparable with those of M. mulatta and M. nigra. Histology of islets is similar to that of humans. Ovoid cell collections of islet cells are scattered throughout the pancreas. Ultrastructure of A, B and D cells is similar to humans. These findings suggest that this relatively underutilized macaques may be a suitable model for biomedical research.     

Apoptotic Mode of Cell Death in Substantia Nigra Following Intranigral Infusion of the Parkinsonian Neurotoxin,MPP<Superscript>+</Superscript> in Sprague-Dawley Rats: Cellular,Molecular and Ultrastructural Evidences

The potent parkinsonian neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is known to cause dopaminergic neurodegeneration in nigrostriatal system. In the present study we investigated the nuclear morphology of cells in the substantia nigra pars compacta (SNpc) region of rats following unilateral intranigral infusion of the active metabolite, 1-methyl-4-phenyl pyridinium ion (MPP⁺), which resulted in a dose-dependent and prolonged dopamine depletion in the ipsilateral striatum. There appeared a substantial loss of tyrosine hydroxylase immunoreactive neurons in the SNpc that received the neurotoxin. Specific nuclear staining with Hoechst 33342 or acridine orange revealed bright pyknotic, shrunken, distorted nuclei and condensed chromatin with perinuclear nucleolus respectively following visualization with the former and latter dyes in the ipsilateral SNpc, as compared to the round, intact nuclei and centrally positioned nucleolus in the contralateral side. Ultrastructural details of the nucleus under transmission electron microscope confirmed distorted nuclear organization with shrunken or condensed nuclei and disrupted nuclear membrane. These features are typical of nucleus undergoing apoptosis, and suggest that MPP⁺ causes dopaminergic neuronal death through an apoptotic mode. Typical laddering pattern of genomic DNA isolated from the ipsilateral SN in agarose gel electrophoresis conclusively established apoptosis following intranigral administration of MPP⁺ in rats. Rebecca Banerjee and Sen Sreetama contributed equally to this paper.     

Structural basis for the role of Asp-120 in metallo-beta-lactamases

Metallo-beta-lactamases (mbetals) are zinc-dependent enzymes that hydrolyze a wide range of beta-lactam antibiotics. The mbetal active site features an invariant Asp-120 that ligates one of the two metal ions (Zn2) and a metal-bridging water/hydroxide (Wat1). Previous studies show that substitutions at Asp-120 dramatically affect mbetal activity, but no consensus exists as to its role in beta-lactam turnover. Here we present crystal structures of the Asn and Cys mutants of Asp-120 of the L1 mbetal from Stenotrophomonas maltophilia. Both mutants retain a dinuclear zinc center with Wat1 present. In the essentially inactive Cys enzyme Zn2 is displaced to a more buried position relative to that in the wild-type enzyme. In the catalytically impaired Asn enzyme the coordination of Zn2 is altered, neither it nor Wat1 is coordinated by Asn-120, and the N-terminal 19 amino acids, important to cooperative interactions between subunits in the wild-type enzyme, are disordered. Comparison with the structure of L1 complexed with the hydrolyzed oxacephem moxalactam suggests that in the Cys mutant Zn2 can no longer make stabilizing interactions with anionic nitrogen species formed in the hydrolytic reaction. The diminished activity of the Asn mutant arises from a combination of loss of intersubunit interactions and impaired proton transfer to, and reduced interaction of Zn2 with, the substrate amide nitrogen. We conclude that, while interactions of Asp-120 with active site water molecules are important to proton transfer and possibly nucleophilic attack by Wat1, its primary role is to optimally position Zn2 for catalytically important interactions with the charged amide nitrogen of substrate.