Christopher Stubbs - US grants

It is proposed to determine the effects of ethanol on protein-lipid interactions in membranes and how lipids modified as a result of chronic ethanol ingestion might modulate the effects. The first aspect we propose to investigate is the binding and penetration of proteins which spontaneously incorporate into membranes. We will determine how this is affected by ethanol and whether is differs for lipids from membranes of ethanol tolerant animals as compared to controls using cytochrome b5 and apocytochrome C. The second aspect of protein-lipid interaction. This region has been difficult to study in the past but we have developed a new approach which in effect spectroscopically isolates this region, using time resolved fluorescence. We propose to use this approach to investigate the effect of ethanol perturbation on this region and whether it differs for different proteins. We will use cytochrome b5, apocytochrome C, gramicidin and glycophorin, as relatively simple representative membrane proteins. We will also determine whether the protein-lipid interfacial region differs in protein reconstituted with lipids from ethanol-tolerant animals. The functional significance of ethanol perturbation of the protein-lipid interfacial region will be explored using skeletal muscle sarcoplasmic reticulum calcium ATP'ase.

It is proposed to pursue the hypothesis that functional effects at the level of the cell membrane, brought about by ethanol, and by lipids that are modified as a result of chronic-ethanol ingestion, may be produced by action at the membrane protein-lipid interface. In the past three years of support several approaches to test the hypothesis have been developed. So far we have found that the adaptation to ethanol-ingestion, consisting of development of a resistance to perturbation by ethanol after chronic-ethanol ingestion-or membrane tolerance, is expressed at the protein-lipid interface. It is now proposed to determine the underlying mechanism and its physical and compositional basis. It is also proposed to pursue results that indicate that ethanol perturbs hydrogen bonding and hydration levels at the protein-lipid interface. We also plan to determine the connection between these effects and altered levels of phospholipid unsaturation that result from the effects of chronic-ethanol ingestion, that preliminary work suggests may important. Many hydrophobic compounds, including ethanol, are classed as general anesthetics and we have found that some more potent compounds were found to interact at the protein-lipid interface, with an apparently higher affinity than would be predicted by hydrophobicity or membrane lipid bilayer partitioning properties. It is postulated that the mechanism is due to an interference with hydrogen bonding. It is therefore proposed to verify this mechanism and to determine if it extends to alcohols and ethanol. The studies will be pursued with natural and model membranes using various fluorescence and other spectroscopic approaches.

9512594 Stubbs Silicon technology has revolutionized astronomy by giving us near perfect CCD light detectors that have very high quantum efficiency and linearity compared to the old standard: photographic plates. However, individual CCD detectors are very small compared to photographic plates and cover only a small amount of sky so that most of the light gathered by the telescope mirror is wasted. This program will develop a powerful wide-field CCD camera system for the Apache Point Observatory (APO) 3.5 meter telescope that will be as large as any yet built (67 million pixels), and will have state-of- the-art read-outs and data processing capability. This versatile instrument will make possible a wide range of astrophysical observing programs, particularly those that attack pressing open questions in observational cosmology such as: How fast is the universe expanding?; Will this expansion continue forever?; what is the nature and distribution of the dark matter that is the gravitationally dominant component of the universe?; and What is the role of particle physics in the birth and evolution of the universe? ***

APPLICANT'S ABSTRACT: It is proposed to investigate the hypothesis that the function of the family of related signaling proteins that translocate from the cytosol to the liver plasma membrane is modified as a result of chronic ethanol ingestion. These proteins, which include protein kinase C (PKC), phospholipase A2 (PLA2), diacylglycerol kinase, rafkinase, phospholipase Cy etc., require phosphati-dylserine and calcium for the translocation, have homology in Ca2+ -binding and/or zinc finger domain regions and are implicated in chronic ethanol induced effects on signal transduction. Preliminary and published data indicates that both PKC and PLA2 activities are modified as a result of chronic ethanol-induced lipid alteration in a manner consistent with the hypothesis. It has been established from our previous studies that: (a) the effect on PLA2 is due to a modification primarily in phosphatidylserine (PS) and (b) that the modification consists of an increase in fatty acyl unsaturation (increased docosahexaenoyl (n-3 22:6) and decreased in arachidonyl (n-6 20:4) containing PS-molecular species). Preliminary studies indicate that PKC, which requires PS for activity, is highly sensitive to PS-unsaturation, determined using highly purified-recombinant PKC and defined lipid bilayer vesicle systems. Further studies are proposed to more closely define (a) which of the Ca2+ -dependent and -independent PKC molecular species that occur in the liver cell are affected. The type and amount of each PKC isoform that associates with liver plasma membranes from livers of rats fed ethanol chronically will be determined along with and the PS-unsaturation. A lipid test system will be developed and used as a model for further studies to determine if the effects of chronic ethanol induced modifications interfere with PKC-membrane association and PKC activator-interaction using recently published methodology. Finally, we will explore the possibility that chronic ethanol-feeding affects the range of signaling proteins that translocate to the membrane in a PS dependent manner thus providing a mechanism that may underlie many of the effects of chronic ethanol ingestion on liver cell signal transduction.

9800028 Stubbs The MACHO project uses the principle of gravitational microlensing to search for compact objects in the halo, disk, and bulge of our Galaxy. The experiment has been acquiring data since 1992, at the Mt. Stromlo Observatory in Australia. The main objective of the project is to detect and/or constrain the existence of compact objects (MACHOS) in the dark matter halo of our Galaxy. The search to date has been very successful and has made important progress toward determining the nature of the Galaxy's dark matter halo. Dr. Christopher Stubbs at the University of Washington and his colleagues will continue this project for another three year period.

9731862 STUBBS Professor Christopher Stubbs and others at the University of Washington, in collaboration with researchers at Mt. Stromlo Observatory, Australia, are engaged in a search for supernovae (SNe) in dense clusters of galaxies. This will contribute to our understanding of the geometry of the universe, to our understanding of distortions in the expanding universe, and to our knowledge of the final stages of stellar evolution. The U.S. group has 5% of the time on the MaCHO 1.3m telescope and wide-field imaging camera, an instrument that is essential to the success of the project, located at Mt. Stromlo, Australia. This award will support the travel to Mt. Stromlo and per diem of Professor Christopher Stubbs' graduate student, David Reiss, for three trips a year for two years during which he will be working on his Ph.D thesis. The Australian counterpart is Dr. Brian P. Schmidt of the Institute for Advanced Studies, Mt. Stromlo and Siding Spring Observatories, Australia.

9816291
STUBBS

Professor Christopher Stubbs and others at the University of Washington, in collaboration with researchers at Mt. Stromlo Observatory, Australia, are engaged in a search for supernovae (SNe) in dense clusters of galaxies. This will contribute to our understanding of the geometry of the universe, to our understanding of distortions in the expanding universe, and to our knowledge of the final stages of stellar evolution. The U.S. group has 5% of the time on the MaCHO 1.3m telescope and wide-field imaging camera, an instrument which is essential to the success of the project, located at Mt. Stromlo, Australia. This award will support the travel to Mt. Stromlo and per diem of Professor Christopher Stubbs' graduate student, David Reiss, for three trips a year for two years during which he will be working on his Ph.D thesis. The Australian counterpart is Dr. Brian P. Schmidt of the Institute for Advanced Studies, Mt. Stromlo and Siding Spring Observatories, Australia.

Protein kinase C [PKC] is a key regulatory element in synaptic signal transduction. During the previous period of support it was found that alcohols and general anesthetics inhibit PKC with a potency that is a linear function of the octanol/buffer partition coefficient indicating a hydrophobic binding site. The effect follows the Meyer-Overton "rule" of alcohol and anesthetic action which requires that anesthetic effects be a linear function of hydrophobicity. The measurements were made in the absence of membrane lipids suggesting the site is on the molecule itself. This observation has also been obtained at the single isoform level with purified recombinant PKCalpha. Evidence obtained so far points to the phorbol ester binding site as the site of alcohol interaction. While membrane lipid-associated PKCalpha is also inhibited by short chain alcohols, by contrast, long chain alcohols and general anesthetics potentiate activity. To further understand the effects of alcohols and general anesthetics on PKC and its role in ethanol intoxication and general anesthesia it is proposed to extend and broaden the study to encompass the three major PKC isoforms classes since they possess widely differing properties. Also since PKC has been found to target cytoskeletal elements involved in neurotransmission, we propose to investigate this process following preliminary findings showing ethanol effects. To accomplish this the cDNA encoding the major PKC isoforms that occur in brain, has been obtained and inserted into baculovirus vectors and the PKC expressed in Sf9 cells and purified. It is proposed to investigate the effect of alcohols with respect to activator-and substrate-type differences that have been observed in preliminary experiments. The site of alcohol interaction within PKC and its molecular details will be determined using a series of full length and fusion-protein mutants along with the effects of alcohols on the increased proteolytic sensitivity of activated-PKC that leads to down regulation. Overall the results should contribute to understanding of the mechanism and consequences of ethanol and general anesthetics on synaptic signaling and neurotransmission.

It is proposed to investigate the hypothesis that ethanol resides at sites on the hydrophobic surface and in inter-helical regions of trans-membrane proteins where it modifies the ability of the protein to change its conformation as part of normal functioning. It is proposed that "sites" of interaction may be quite common, although few are likely to provide a significant enough interaction to modulate function. The ethanol interaction partly involves hydrophobic interactions but in addition a weakening of a protein-lipid hydrogen bonding network that include water molecules at sensitive regions on the protein is proposed to play a major role. Published work from this laboratory on lipid bilayer membranes with model membrane proteins inserted indicate that ethanol significantly perturbs the phospholipid head group, acyl chain and protein-lipid interfacial regions in membranes by weakening the hydrogen bond network. In addition evidence that chronic-ethanol ingestion leads to adaptive changes in the cell membrane lipids that attenuate this (direct) effect of ethanol has been presented. In this renewal it proposed to test the hypothesis of a link between an ethanol-induced perturbation of the hydrogen bond network and effects on function in seven-transmembrane G-protein coupled receptors (GPCR) which are a large family of proteins that initiate numerous signal transduction cascades in cells. The cDNA for two representative GPCR: the vasopressin-V1A and beta2-adrenergic receptors has been inserted into baculovirus vectors and expressed in Sf9 insect cells and will be affinity purified and reconstituted into defined lipids. The reconstitution will include lipids possessing the chronic ethanol-induced modification, to test the role of perturbation the hydrogen bond network on the agonist- induced trans-membrane inter- and intra-helical conformational changes that leads to G-protein activation. Putative regions that might harbor ethanol "sites" and that would be likely to lead to functional perturbation will be further delineated using point mutations.

It is proposed to investigate the hypothesis that ethanol resides at sites on the hydrophobic surface and in inter-helical regions of trans-membrane proteins where it modifies the ability of the protein to change its conformation as part of normal functioning. It is proposed that "sites" of interaction may be quite common, although few are likely to provide a significant enough interaction to modulate function. The ethanol interaction partly involves hydrophobic interactions but in addition a weakening of a protein-lipid hydrogen bonding network that include water molecules at sensitive regions on the protein is proposed to play a major role. Published work from this laboratory on lipid bilayer membranes with model membrane proteins inserted indicate that ethanol significantly perturbs the phospholipid head group, acyl chain and protein-lipid interfacial regions in membranes by weakening the hydrogen bond network. In addition evidence that chronic-ethanol ingestion leads to adaptive changes in the cell membrane lipids that attenuate this (direct) effect of ethanol has been presented. In this renewal it proposed to test the hypothesis of a link between an ethanol-induced perturbation of the hydrogen bond network and effects on function in seven-transmembrane G-protein coupled receptors (GPCR) which are a large family of proteins that initiate numerous signal transduction cascades in cells. The cDNA for two representative GPCR: the vasopressin-V1A and beta2-adrenergic receptors has been inserted into baculovirus vectors and expressed in Sf9 insect cells and will be affinity purified and reconstituted into defined lipids. The reconstitution will include lipids possessing the chronic ethanol-induced modification, to test the role of perturbation the hydrogen bond network on the agonist- induced trans-membrane inter- and intra-helical conformational changes that leads to G-protein activation. Putative regions that might harbor ethanol "sites" and that would be likely to lead to functional perturbation will be further delineated using point mutations.

AST 0073845
Alcock

The exploration of the outer Solar System has developed rapidly in the past decade, with the discovery of large numbers of small, solid objects in the trans-Neptunian region. These objects have been found with CCD cameras on medium and large sized telescopes, which to date have surveyed ~100 square degrees of sky. This new project, led by Dr. Charles Alcock of the University of Pennsylvania, is intended to provide an important complement to recent, very successful surveys of the outer Solar System. Specifically, Dr. Alcock's survey will cover ~3000 square degrees with high sensitivity to objects in the size range between Pluto and recently discovered trans-Neptunian objects (TNO). A powerful observing system developed for the MACHO project (a project to detect massive objects via the technique of gravitational lensing) will be utilized for the survey. This award is made through the Planetary Astronomy Program.
***

AST-0206329
Stubbs
A recent convergence of observational results has led cosmologists to the surprising conclusion that not only is the Universe expanding, but that the rate of expansion is increasing. This can be thought of as anti-gravity between regions of the vacuum. It appears that the Universe at present is dominated by the gravitational effect of this mysterious Dark Energy. Distant supernovae (exploding stars) provide a means to map out the history of the expansion of the Universe, and can be used to distinguish between different scenarios for the physics that drives an accelerating expansion. This project will detect and monitor over 200 supernovae in order to test different hypotheses for the nature of the Dark Energy.

enzyme activity; protein transport; protein kinase C; membrane activity; liver metabolism; biological signal transduction; ethanol; intracellular transport; intermolecular interaction; protein signal sequence; protein isoforms; fatty acid metabolism; membrane model; fatty acids; membrane lipids; phosphatidylserines; membrane transport proteins; lipid bilayer membrane; lipid metabolism; membrane proteins; confocal scanning microscopy; nuclear magnetic resonance spectroscopy; laboratory rat; fluorescence spectrometry; electron spin resonance spectroscopy;

AST-0507475
Stubbs

One of today's most pressing open questions in physics concerns the nature of the dark energy driving the accelerating expansion of the Universe. Although there is currently little doubt that dark energy represents some new and fundamental physics, there is still a need to distinguish between various possibilities for its nature: is it gravitational, residing in Einstein's cosmological constant, or exotic matter, coming from quantum mechanical processes in the vacuum? This project continues an ambitious survey, currently about half complete, to use some 200 Type Ia supernovae between redshifts of 0.2 and 0.8 to map out the expansion history of the Universe. It should be able to constrain a critical dark energy parameter with a fractional uncertainty of only 10%.

Many undergraduate and graduate students have been involved in this cutting-edge research project, and the enhanced capabilities it required at an NSF-supported telescope have benefited all users of that facility. Since the nature of the dark energy is a mystery that has captivated the imagination of the general public, project members will continue to be very engaged in both formal and informal educational activities on this topic.

AST-0506752
Stubbs

One of today's most pressing open issues is to understand the nature of the dark energy apparently responsible for the accelerating expansion of the Universe. This project will carry out a one hundred square degree multi-band optical survey of the southern sky, in conjunction with even deeper images of smaller fields, coordinated with a millimeter-wave survey that will map the cosmic microwave background (CMB) to detect galaxy clusters out to moderate redshifts. These coordinated surveys will enable the most precise cosmological study to date with galaxy clusters, and will enable complementary studies of the dark energy using supernova distances, cosmic shear, and the galaxy power spectrum. This research will use the NSF-supported Blanco telescope at the Cerro Tololo Interamerican Observatory, and the privately supported Magellan telescope, both operational in Chile, and the CMB data will come from the NSF-funded South Pole Telescope, currently under construction.

The key science question of the dark energy has already engaged significant public interest. This research will leverage the scientific productivity of multiple NSF-funded facilities, break new ground in real-time image analysis and observing optimization, directly benefit future large surveys currently in the planning stages, and continue a tradition of graduate and undergraduate student participation.

This project centers on optical (visible-light) observations using large, ground-based telescopes, designed to maximally exploit the science return from the South Pole Telescope (SPT). The SPT, an NSF-funded experiment at the Amundsen-Scott South Pole Station, consists of a 10-meter diameter, millimeter-wave radio telescope designed to measure the subtle fluctuations in the brightness of the cosmic background radiation (CBR) at multiple wavelengths. As part of its survey operations, the SPT is producing a list of possible galaxy clusters detected by the effect the hot, intra-cluster plasma has on the CBR. The size of this spectral distortion, known as the Sunyaev-Zel'dovich (SZ) effect, is independent of distance, offering the opportunity to inventory galaxy clusters in the universe, relatively free of the strong distance bias that affects other types of observations. In this project the collaborating team (from Smithsonian Astrophysical Observatory and Harvard University) will perform optical follow-up observations of the SPT cluster candidates, to confirm the existence of the clusters, to obtain their redshifts, and to identify the member galaxies. The team will make use of an array of existing instrumentation on the current suite of large telescopes, including PISCO, a multi-bandpass imaging camera developed with NSF support for the 6.5-meter Magellan telescope. The primary result of this work will be an unprecedented, volume-limited catalog of approximately 600 galaxy clusters. The full catalog of photometric and spectroscopic observations will be released to the astronomical community through a web-accessible archive using well-developed database architectures. These observations are intended to enable a wide variety of subsequent inquiries, including studies of galaxy evolution in different environments, efforts to further constrain cosmological parameters, and cluster mass measurements through gravitational lensing. The project will support the work of a postdoctoral fellow, as well as graduate and undergraduate students, and will be publicized through the Principal Investigators' close involvement with significant public-media projects.