Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Novel Antileukemic Compound Ingenol 3-Angelate Inhibits T Cell Apoptosis by Activating Protein Kinase C��

Members of the protein kinase C (PKC) family of serine-threonine kinases are important regulators of immune cell survival. Ingenol 3-angelate (PEP005) activates a broad range of PKC isoforms and induces apoptosis in acute myeloid leukemia cells by activating the PKC isoform PKCδ. We show here that, in contrast to its effect on leukemic cells, PEP005 provides a strong survival signal to resting and activated human T cells. The antiapoptotic effect depends upon the activation of PKCθ. This PKC isoform is expressed in T cells but is absent in myeloid cells. Further studies of the mechanism involved in this process showed that PEP005 inhibited activated CD8⁺ T cell apoptosis through the activation of NFκB downstream of PKCθ, leading to increased expression of the antiapoptotic proteins Mcl-1 and Bcl-x_L. Transfection of CD8⁺ T cells with dominant-negative PKCθ diminished the prosurvival effect of PEP005 significantly. Ectopic expression of PKCθ in the acute myeloid leukemia cell line NB4 turned their response to PEP005 from an increased to decreased rate of apoptosis. Therefore, in contrast to myeloid leukemia cells, PEP005 provides a strong survival signal to T cells, and the expression of functional PKCθ influences whether PKC activation leads to an anti- or proapoptotic outcome in the cell types tested.     

Genome sequence of the obligate methanotroph Methylosinus trichosporium strain OB3b

Methylosinus trichosporium OB3b (for "oddball" strain 3b) is an obligate aerobic methane-oxidizing alphaproteobacterium that was originally isolated in 1970 by Roger Whittenbury and colleagues. This strain has since been used extensively to elucidate the structure and function of several key enzymes of methane oxidation, including both particulate and soluble methane monooxygenase (sMMO) and the extracellular copper chelator methanobactin. In particular, the catalytic properties of soluble methane monooxygenase from M. trichosporium OB3b have been well characterized in context with biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of the M. trichosporium OB3b genome is the first reported from a member of the Methylocystaceae family in the order Rhizobiales.     

A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.     

Evaluating a Large‐Scale Eelgrass Restoration Project in the Chesapeake Bay

Approximately 90,000 shoots of eelgrass (Zostera marina) were planted over 3 years (2003–2005) at Piney Point (PP) in the lower Potomac River estuary in the Chesapeake Bay (mid‐Atlantic coast of North America) following 3 years of habitat evaluation using a Preliminary Transplant Suitability Index (PTSI) and test plantings. Initial survival was high for the 2003 and 2004 plantings; however, most of the eelgrass died during the summer following the fall planting. Habitat quality and restoration success were monitored for the 2005 plantings and compared to a nearby restoration site (St. George Island [SGI]). Eelgrass planted at PP in the fall of 2005 declined through the summer of 2006 with some recovery in the spring of 2007, but was gone by the end of the summer of 2007. The summer decline from late July to mid‐August of 2006 coincided with water temperatures greater than 30°C, hypoxic oxygen (0–3 mg/L) concentrations, and low percent light at leaf level (PLL < 15%). Epiphyte loads were much heavier at PP than at SGI, despite similar water quality. We suggest that this was the result of higher wave exposure at PP. All of these factors are likely to have contributed to the mortality of the 2005 plantings. Submerged aquatic vegetation habitat quality based on the PTSI, median PLL during the growing season, and test plantings did not explain the decline of the plantings. Restoration site selection criteria should be expanded to include the effects of wave exposure on self‐shading and epiphyte loads, and the potential for both short‐term exposures to stressful conditions and long‐term changes in habitat quality.     

Quantification of cortisol and 6 beta-hydroxycortisol in human urine by LC-MS/MS,and gender-specific evaluation of the metabolic ratio as biomarker of CYP3A activity

Drug–drug and food–drug interactions are often due to an inhibition or induction of drug-metabolizing cytochrome P450 (CYP) enzymes and may result in non-response or adverse reactions. Hence, phenotypic biomarkers of CYP activity appear as useful tools for individualized pharmacotherapy. The metabolic ratio (MR) of the concentration of 6β-hydroxycortisol (6β-OHC) to cortisol (MR 6β-OHC/cortisol) in human urine had been proposed as an endogenous marker for CYP3A activity. Here, we report on the improvement of published LC-MS/MS methods for the simultaneous quantification of cortisol and 6β-OHC, using on-line sample cleanup by column switching and isotope-labeled analogues as internal standards. [²H₂]6β-OHC was prepared by incubation of human recombinant CYP3A4 with commercially available [²H₂]cortisol. Analytical sensitivity could be increased about 10-fold. The first morning urine of 69 female and 27 male healthy volunteers was analyzed for cortisol and 6β-OHC. Concentrations ranged from 1.0 to 142 and 24 to 670 ng/mL, respectively. Individual MR 6β-OHC/cortisol varied more than 20-fold and we were able to show for the first time for a Caucasian population significantly higher MR values in females as compared to males. This non-invasive biomarker for CYP3A activity lends itself for the study of genetic differences as well as enzyme induction or inhibition in the clinical setting without the need of using a probe drug.     

Evaluation of extraction methods for use with NMR-based metabolomics in the marine polychaete ragworm, Hediste diversicolor

The sediment-dwelling polychaete, Hediste diversicolor, is commonly found in Northern temperate estuaries. Its limited mobility and tolerance to polluted conditions makes it a good candidate for biological monitoring. Moreover, its importance in the functioning of the sediment ecosystem has caused it to be described as a keystone species. Here we present the development of analytical methodology that will enable the use of H. diversicolor in environmental metabolomics studies for the biomonitoring of estuarine ecosystems. Polar and non-polar extraction solvents have been used to solubilise a wide range of metabolites. Extraction solvents assessed include: aqueous phosphate buffer solution, methanol:chloroform:water (1:1:0.9), methanol:water (1:1 and 2:1) and chloroform. The metabolites were analysed using 1-dimensional (1D) ¹H nuclear magnetic resonance (NMR) spectroscopy. Using the methanol:water (1:1) method, previous freezing to aid cell rupture did not result in an enhanced extraction. Removal of methanol with a speed vacuum resulted in reduction in yield. Methanol:water (1:1) and chloroform extractions proved to be the most appropriate techniques based on the sample yield and repeatability. NMR-based metabolomics in the ragworm can now be used to understand the ecophysiology of this important estuarine organism and has applications in biomonitoring, biomarker development and ecotoxicological studies.     

百脉根BIO和豌豆突变位点ELE2的比较基因组定位(英文)

豆科两侧对称花的花瓣具有背腹(DV)的分化以及可变的器官内部(IN)非对称性,在大小与形状上显示出不同的发育特征;因而花瓣的发育为克隆决定植物器官的形状与大小的关键基因提供了很好的实验系统。本研究对百脉根中BIO基因进行研究。百脉根bio突变体具有多效性,既影响花器官内部的对称性也影响器官的大小和育性,豌豆ele突变体的表型与bio相似。定位结果表明BIO和ELE2位于豆科基因组的共线性区段,提示BIO和ELE2可能是同源基因突变所致。本研究利用比较基因组定位方法,将BIO和ELE2候选基因锚定在豆科模式植物百脉根和蒺藜苜蓿基因组含有11个同源基因的BAC重叠群上。BIO和ELE2基因的克隆将有助于揭示豆科花瓣形态和大小调控的分子机理,进而为豆科作物遗传改良提供分子理论基础。