Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

Dynamics of the phosphoinositide 3-kinase p110δ interaction with p85α and membranes reveals aspects of regulation distinct from p110α

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.     

Federal Agency biodefense funding, FY2011-FY2012

Since 2001, the United States government has spent substantial resources on preparing the nation against a bioterrorist attack. Earlier articles in this series have analyzed civilian biodefense funding by the federal government for fiscal years (FY) 2001 through proposed funding for FY2011. This article updates those figures with budgeted amounts for FY2012, specifically analyzing the budgets and allocations for biodefense at the Departments of Health and Human Services, Defense, Homeland Security, Agriculture, Commerce, and State; the Environmental Protection Agency; and the National Science Foundation. This article also includes an updated assessment of the proportion of biodefense funding provided for programs that address multiple scientific, public health, healthcare, national security, and international security issues in addition to biodefense. The FY2012 federal budget for civilian biodefense totals $6.42 billion. Of that total, $5.78 billion (90%) is budgeted for programs that have both biodefense and nonbiodefense goals and applications, and $637.6 million (10%) is budgeted for programs that have objectives solely related to biodefense.     

A holistic approach to marine eco-systems biology

The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.     

A transmembrane glycoprotein, gp38, is a novel marker for immature hepatic progenitor cells in fetal mouse livers

Previously, we clarified the surface antigen profiles of hepatic progenitor cells (HPCs) in fetal liver tissue as the CD49f(+)CD45(-)Thy1(-) cell fraction. However, these cells were a heterogeneous cell population containing various stages of differentiation. This study aimed to detect more immature HPCs, using a novel surface antigen, gp38. After the collagenase digestion of fetal livers harvested from E13.5 to E18.5 fetal mice, HPCs were obtained and divided into two subpopulations using flow cytometry: gp38-positive HPCs, and gp38-negative HPCs. Both types of HPCs were characterized by immunocytochemistry and RT-PCR. The proliferative activity was compared by BrdU incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay. Furthermore, the comprehensive gene expression was investigated by DNA microarray. Both types of HPCs expressed alpha-fetoprotein. However, the gp38-positive HPCs derived from E13.5 fetal livers did not express albumin or cytokeratin 19, while the gp38-negative HPCs did. DNA microarray revealed that some genes related to the Wnt signal pathway were up-regulated in the gp38-positive HPCs. Furthermore, Wnt3a had a proliferative effect on the gp38-positive HPCs. In conclusion, the gp38-positive HPCs derived from fetal liver tissue until E13.5 could therefore be candidates for hepatic stem cells in the fetal liver.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

The CRIT framework for identifying cross patterns in systems biology and application to chemogenomics

Biological data is often tabular but finding statistically valid connections between entities in a sequence of tables can be problematic - for example, connecting particular entities in a drug property table to gene properties in a second table, using a third table associating genes with drugs. Here we present an approach (CRIT) to find connections such as these and show how it can be applied in a variety of genomic contexts including chemogenomics data.     

Mosquito host selection varies seasonally with host availability and mosquito density

Host selection by vector mosquitoes is a critical component of virus proliferation, particularly for viruses such as West Nile (WNV) that are transmitted enzootically to a variety of avian hosts, and tangentially to dead-end hosts such as humans. Culex tarsalis is a principal vector of WNV in rural areas of western North America. Based on previous work, Cx. tarsalis utilizes a variety of avian and mammalian hosts and tends to feed more frequently on mammals in the late summer than during the rest of the year. To further explore this and other temporal changes in host selection, bloodfed females were collected at a rural farmstead and heron nesting site in Northern California from May 2008 through May 2009, and bloodmeal hosts identified using either a microsphere-based array or by sequencing of the mitochondrial cytochrome c oxidase I (COI) gene. Host composition during summer was dominated by four species of nesting Ardeidae. In addition, the site was populated with various passerine species as well as domestic farm animals and humans. When present, Cx. tarsalis fed predominantly (>80%) upon the ardeids, with Black-crowned Night-Herons, a highly competent WNV host, the most prevalent summer host. As the ardeids fledged and left the area and mosquito abundance increased in late summer, Cx. tarsalis feeding shifted to include more mammals, primarily cattle, and a high diversity of avian species. In the winter, Yellow-billed Magpies and House Sparrows were the predominant hosts, and Yellow-billed Magpies and American Robins were fed upon more frequently than expected given their relative abundance. These data demonstrated that host selection was likely based both on host availability and differences in utilization, that the shift of bloodfeeding to include more mammalian hosts was likely the result of both host availability and increased mosquito abundance, and that WNV-competent hosts were fed upon by Cx. tarsalis throughout the year.     

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.Download video file.^{(60M, mov)}