Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins

Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy

AbstractThe intrinsic fluorescence properties of the oncogene protein p21N-ras, p21H-ras and one of its transforming mutants, p21N-ras (Va1112), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect

Distribution Pattern Variability of Granular VRT Applicators

Granular applicators equipped with variable-rate technology (VRT) have gained popularity in recent years as a result of increased interest in variable-rate application. The purpose of this investigation was to characterize distribution patterns at varying rates for different granular applicators. Uniform-rate (UR) tests were conducted to assess the accuracy of variable-rate application from four granular applicators: two spinner-disc spreaders (A and B), and two pneumatic applicators (C and D). Pattern results indicated a consistent triangular pattern for spinner spreader B and consistent patterns for the pneumatic applicators (C and D). However, applicator D produced pattern variations at the center and right side. Simulated overlap analysis generated CVs \u3c 20% for applicators B and C. Applicator A performed well at the two lower rates (CVs \u3c 19%) but not at the highest rate (CV = 27%). Pattern unevenness for applicator D produced CVs between 25% and 34%. The spinner-disc spreaders over-applied, while the pneumatic applicators under-applied at the margins, suggesting an adjustment to the effective swath spacing or spinner-disc speed is needed to improve application accuracy. Further, overlap plots indicated pattern variability even when acceptable CVs were attained for applicators B and C. Therefore, it is recommended that CVs accompany simulated overlap pattern plots to ensure proper calibration of VRT equipment. Swath spacing analysis indicated that three of the four applicator spacings could be changed from the recommended value to improve application uniformity. Pattern comparisons showed that pattern shifts occurred for applicator A (P = 0.0092) with increasing application rate but not for applicators B, C, and D. These results demonstrate potential application errors with VRT and the need for proper calibration to maintain acceptable performance. Further, this investigation demonstrates the need for a VRT equipment testing standard

The Kondo Dynamics of YbIn(1-x)AgxCu4

We present an infrared/optical study of the dynamics of the strongly correlated electron system YbIn(1-x)AgxCu4 as a function of doping and temperature for x ranging from 0 to 1, and T between 20 and 300 K. This study reveals information about the unusual phase transition as well as the phases themselves. Scaling relations emerge from the data and are investigated in detail using a periodic Anderson model based calculation. We also provide a picture in which to view both the low and high-energy x-dependent features of the infrared data, including identification of high energy, temperature dependent features.Comment: 12 pages, 11 figures, submitted Phys. Rev.

Gateway Hosting at Indiana University

In: Proceedings of the TeraGrid 2009 Conference, 22-25 June, Arlington, VA.The gateway hosting service at Indiana University provides science gateways and portals with hosting resources to facilitate the use of computation resources and storage within the TeraGrid. This service is designed with high availability in mind and is deployed across the Indianapolis and Bloomington campuses with redundant network, power, and storage. The service uses OpenVZ to give each gateway or portal its own virtual environment while making the most efficient use of the hardware and administrative resources. OpenVZ’s user beancounter quota system and fair-share scheduling for processes and I/O allows fair distribution of resource between virtual machines while allowing full utilization of the hardware. The ability to do live migration allows kernel updates without service interruption. Indiana University’s research network provides multiple low latency high bandwidth connections between campuses, other TeraGrid resource providers, and the Internet at large. The service is in use by a variety of projects such as FlyBase and TeraGrid Information Services and, since the service was put into production in August 2008, there have been 5.37 hours of down time

Linear Optics Design for PS2

The design considerations and key parameters for the replacement of the CERN Proton Synchrotron (PS) with a new ring (PS2), as part of the upgrade of the LHC injector complex are summarized. Classical linear optics solutions including standard FODO, doublet and triplet cells with real transition energy, are studied. Particular emphasis is given to the tuning and optimisation of Negative Momentum Compaction (NMC) cells with imaginary transition energy. The optics of the high energy transfer line is also presented

Growing and Marketing Watermelons.

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Implications of dealing with airborne substances and reactive oxygen species: what mammalian lungs, animals, and plants have to say?

A gas-exchange structure interacts with the environment and is constantly challenged by contaminants that may elicit defense responses, thus compromising its primary function. It is also exposed to high concentrations of O2 that can generate reactive oxygen species (ROS). Revisiting the lung of mammals, an integrative picture emerges, indicating that this bronchi-alveolar structure deals with inflammation in a special way, which minimizes compromising the gas-exchange role. Depending on the challenge, pro-inflammatory or antiinflammatory responses are elicited by conserved molecules, such as surfactant proteins A and D. An even broader picture points to the participation of airway sensors, responsive to inflammatory mediators, in a loop linking the immunological and nervous systems and expanding the role played by respiratory organs in functions other than gas-exchange. A byproduct of exposure to high concentration of O2 is the formation of superoxide (), hydrogen peroxide (H2O2), hydroxyl radical (HO•), and other ROS, which are known to be toxic to different types of cells, including the lung epithelium. A balance between antioxidants and oxidants exists; in pulmonary epithelial cells high intracellular and extracellular levels of antioxidants are found. Antioxidant adaptations related to plant and animal life-styles involve a broad range of overlapping strategies based on well-conserved molecules. Glutathione (GSH) is an abundant and ubiquitous thiol-tripeptide antioxidant, also present in lungs, whose role in providing information on the intracellular redox state of animals and plants is well established. In these organisms, GSH influences gene expression associated with stress, maximizing defense responses. Several enzymatic antioxidants, such as glutathione peroxidase (GPx), glutathione reductase, glutathione S-transferase, and glucose 6-phosphate dehydrogenase participate in the redox system; in animals that are stress-tolerant GPx is a key element against oxidative assaults. Most importantly, alternative roles of ROS as signaling molecules have been found in all plants and animals. For example, alveolar macrophages produce that act as second messengers, in addition to having a bactericidal role. The nonradical ROS H2O2 signals inflammation in mammalian lungs, apoptosis in different animal tissues, and is also involved in stomatal closure, root development, gene expression, and defense responses of plants. Antioxidant adaptations in some water-breathing animals involve the excretion of H2O2 by diffusion through gas-exchange structures. The fine balance among a multitude of factors and cells makes the difference between damage and protection in animals and plants. Knowledge about the mechanisms and consequences of these molecular interactions is now starting to be integrate