Structural Characterization of the Integrin Αiibβ3 Transmembrane and Cytosolic Domains

Integrin’s are the principal cell surface receptors that link the cytoskeleton to the extracellular matrix. They exist in active conformations that can bind extracellular ligands and resting conformations that cannot. The platelet integrin αIIbβ3 is a prototypical regulated integrin that is resting on a circulating platelet and becomes activated to adhere the platelet to the vascular endothelium or subendothelial matrix. The integrin is composed of α and β subunits and each subunit contains a single transmembrane helix that form an α/β heterodimer in the resting state. Additionally, each subunit contains a cytosolic domain that binds signaling proteins that affect the resting-active equilibrium. Activation signals are transduced across the membrane by separating the transmembrane heterodimer. The structure of the resting integrin αIIbβ3’s transmembrane and cytosolic domains was characterized by molecular modeling and NMR spectroscopy. First, software was developed to model transmembrane helix dimers using experimental mutagenesis results as a modeling restraint. Next, the αIIb/β3 transmembrane heterodimer was modeled and the model was compared to published experimental data and other published models. The model correlated well with experimental findings and converged on the same structure as other top performing models, suggesting this conformation approximates the native interface. The model’s interface includes αIIb residue Met987 and β3 residue Leu712. These residues were mutated to cysteine to crosslink peptides corresponding to the αIIb and β3 cytosolic tails, and the disulfide-linked construct was probed by NMR spectroscopy. NMR revealed that the αIIb and β3 cytosolic tails have a dynamic interface. The αIIb subunit is natively unstructured and the β3 subunit consists of a hydrophobic helix followed by two amphiphilic helices. The amphiphilic portions of β3 include domains that interact with cytosolic proteins, but the membrane embedding of its hydrophobic faces sequesters some of the interacting residues. This result suggests that the integrin’s resting-active equilibrium is coupled to an equilibrium between membrane embedded and solvent exposed conformations of the β3 cytosolic tail, providing new insight into integrin activation

Radon Removal from Potable Water Supplied by Municipal and Single Home Water Wells

Activated charcoal can remove contaminants such as radionuclides from potable water. In northern Virginia, a community water well normally producing about 17 million gallons of water per year plus many small homesite water wells were used to study the monthly and seasonal variation in waterborne radon concentration, These wells were also used to study the ability of activated charcoal to capture the dissolved radon before it reaches the home occupants. It was found that the percentage of radon removal was related to the volume of treated water, the type of activated charcoal, and the length of time that the charcoal was used. In brief, if sufficient activated charcoal was placed in the water treatment tank, the removal of waterborne radon could reach 90 percent. While the intensity of radiation that escaped through the walls of the capture tank was esily detected, estimates indicate that the health risk was minimal while the capture tanks were in operation, and during the replacement of the used and radioactive charcoal

Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea.

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site

Structural organization and interactions of transmembrane domains in tetraspanin proteins

BACKGROUND: Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. RESULTS: Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)(n). In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. CONCLUSION: Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues

Surface Instability in Windblown Sand

We investigate the formation of ripples on the surface of windblown sand based on the one-dimensional model of Nishimori and Ouchi [Phys. Rev. Lett. 71, 197 (1993)], which contains the processes of saltation and grain relaxation. We carry out a nonlinear analysis to determine the propagation speed of the restabilized ripple patterns, and the amplitudes and phases of their first, second, and third harmonics. The agreement between the theory and our numerical simulations is excellent near the onset of instability. We also determine the Eckhaus boundary, outside which the steady ripple patterns are unstable.Comment: 23 pages, 8 figure

Precursors for Atmospheric Plasma-Enhanced Sintering: Low-Temperature Inkjet Printing of Conductive Copper

Bidentate diamine and amino-alcohol ligands have been used to form solid, water-soluble, and air-stable monomeric copper complexes of the type [Cu(NH2CH2CH(R)Y)2(NO3)2] (1, R=H, Y=NH2; 2, R=H, Y=OH; 3, R=Me, Y=OH). The complexes were characterized by elemental analysis, mass spectrometry, infrared spectroscopy, thermal gravimetric analysis, and single-crystal X-ray diffraction. Irrespective of their decomposition temperature, precursors 1–3 yield highly conductive copper features [1.5×10−6Ω m (±5×10−7Ω m)] upon atmospheric-pressure plasma-enhanced sintering

SOCS3 Is a Critical Physiological Negative Regulator of G-CSF Signaling and Emergency Granulopoiesis

AbstractTo determine the importance of suppressor of cytokine signaling-3 (SOCS3) in the regulation of hematopoietic growth factor signaling generally, and of G-CSF-induced cellular responses specifically, we created mice in which the Socs3 gene was deleted in all hematopoietic cells. Although normal until young adulthood, these mice then developed neutrophilia and a spectrum of inflammatory pathologies. When stimulated with G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3 activation and enhanced cellular responses to G-CSF, including an increase in cloning frequency, survival, and proliferative capacity. Consistent with the in vitro findings, mutant mice injected with G-CSF displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed inflammatory neutrophil infiltration into multiple tissues and consequent hind-leg paresis. We conclude that SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and that this is of particular significance during G-CSF-driven emergency granulopoiesis

SOCS1 Is a Critical Inhibitor of Interferon γ Signaling and Prevents the Potentially Fatal Neonatal Actions of this Cytokine

AbstractMice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1−/− mice were shown to exhibit excessive responses typical of those induced by interferon γ (IFNγ), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNγ-dependent capacity to kill L. major parasites. The complex disease in SOCS1−/− mice was prevented by administration of anti-IFNγ antibodies and did not occur in SOCS1−/− mice also lacking the IFNγ gene. Although IFNγ is essential for resistance to a variety of infections, the potential toxic action of IFNγ, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNγ action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses

An Inventory and Assessment of Sample Sources for Survey Research with Agricultural Producers in the U.S.

Researchers need probability samples to collect representative survey data about the behaviors and attitudes of agricultural producers they study in relation to the natural resources that they manage, yet obtaining accurate and complete sampling frames is challenging. We extract data from a publication database to identify the most commonly used sampling frame sources in survey research of agricultural producers in the U.S., finding that government program participant lists are used most often, while private vendor samples are increasingly being purchased. Based on our research experience, we find that for many projects, private vendors can provide the most rigorous samples. Given that survey methods remain a useful and popular method for studying the behaviors and attitudes of producers on a variety of topics, such an assessment and guide is needed for researchers and practitioners

Small businesses and flood impacts: The case of the 2009 flood event in Cockermouth

Flooding can have a devastating impact on businesses, especially on small- and medium-sized enterprises (SMEs) who may be unprepared and vulnerable to the range of both direct and indirect impacts. SMEs may tend to focus on the direct tangible impacts of flooding, limiting their ability to realise the true costs of flooding. Greater understanding of the impacts of flooding is likely to contribute towards increased uptake of flood protection measures by SMEs, particularly during post-flood property reinstatement. This study sought to investigate the full range of impacts experienced by SMEs located in Cockermouth following the floods of 2009. The findings of a questionnaire survey of SMEs revealed that businesses not directly affected by the flooding experienced a range of impacts and that short-term impacts were given a higher significance. A strong correlation was observed between direct, physical flood impacts and post-flood costs of insurance. Significant increases in the costs of property insurance and excesses were noted, meaning that SMEs will be exposed to increased losses in the event of a future flood event. The findings from the research will enable policy makers and professional bodies to make informed decisions to improve the status of advice given to SMEs. The study also adds weight to the case for SMEs to consider investing in property-level flood risk adaptation measures, especially during the post flood reinstatement process