John Douglas Glenton Watson, BSc (Hons I), MB, BS (Hons I), MD (Syd), DPhil (Oxon), FRACP, GAICD - US grants

We have demonstrated that an established Drosophila cell line (Kc cells) is a viable experimental model in which to define sterol independent regulation of HMG-CoA reductase (HMGR) activity. Kc cells modulated HMGR activity in response to mevalonate availability. Furthermore, current evidence suggests that a regulatory signal molecule, for HMGR suppression, was distal to isopentenyll-pyrophosphate (IPP). We observed no simple correlation between HMGR suppression and total IPP flux, total neutral isoprenoid lipid synthesis, or water soluble isoprenoid phosphate ester profile. Kc cells appeared to divert minimally 50% of their mevalonate carbon to n-fatty acids and TCA insoluble end products. Since we do not knot how Kc cells metabolize C10/C15 prenols nor the potential role intermediates from this pathway might play in the regulation of mevalonate synthesis, two related aims were developed for this proposal: (1) to determine if C10/C15 prenol catabolism was linked to mevalonate mediated modulation of HMG-CoA reductase activity and (2) to outline Kc cell C10/C15 prenol catabolism and define selected enzymes of this pathway. In order to demonstrate directly causal linkages between changes in HMG-CoA reductase's functional capacity and the putative post IPP regulatory signal molecule, an additional specific aim was formulated: (3) to utilize a functional, electroporated Kc cell model to identify, directly evaluate putative post IPP regulatory signal molecules, and investigate their mode of action(s). Realization of our Specific Aims, with the Kc model, should provide a clearer understanding of sterol-independent regulation of mevalonate metabolism by eukaryotic cells. Such information will also provide a framework in which to analyze mevalonate mediated regulation of isoprenoid synthesis in the more complex vertebrate cell.

Recent results from this study indicate that noise measurements can reflect not only the product particle size distribution but also the character of the material being broken. Hence, noise parameters could provide valuable fundamental information relating to the differing mechanisms of breakage found in the numerous materials which are ground in ball and rod mills. A better understanding of these mechanisms will assist in the formulation of improved breakage models and result in increased efficiency of present-day grinding operations. To investigate the breakage mechanisms, work is performed on a variety of materials to link particle breakage with overall mill noise levels and with individual particle acoustic emission.

It has been recognized that a major risk factor for coronary heart disease is increased serum cholesterol levels. This finding highlights the importance of achieving a basic understanding, at the molecular level, of the metabolic regulation of isopentenoids in general and cholesterol specifically. The broad aim of this proposal is to assess the role of oxysterol synthesis and/or other isopentenoids in serum (LDL) cholesterol mediated down- regulation of cellular mevalonate synthesis directly and indirectly. We will use a mutant CHO cell line, unable to demethylate 4 alpha-methyl sterols, as our experimental model. The mutant CHO cells' metabolic lesion eliminates the metabolic complexity generated by greater than 20 intermediates between 4,4-dimethyl sterols and cholesterol. As such our changes to detect, isolate, and identify potential regulatory oxysterol(s) synthesized from (3H)mevalonate or preformed (14C/3H)cholesterol are increased. We will pursue a work plan which incorporates the coordinated, kinetic assessment of serum mediated oxysterol synthesis with the onset of the down-regulation for 3-hydroxy, 3-methyl glutaryl coenzyme A (HMG-CoA) reductase/HMG-CoA synthetase activities, and their respective turnover. In addition, we will assess the role of cytochrome P450 dependent processes on the serum mediated, down-regulation cascade. Potential biosynthesized regulatory sterols will be monitored by radio-HPLC, isolated, purified and identified by GC/MS. Radioimmunochemical techniques will be used to assess specific enzyme turnover. We expect our immediate study to yield new information about the obligatory sterologenic events which occur distal to serum lipoprotein uptake and catabolism. Such information should serve as a framework for a new generation of fundamental questions to be addressed with intact animals challenged by a single cholesterol meal. Ultimately we hope that a fundamental knowledge base will be generated to provide the necessary insight for understanding serum mediated control of mevalonate synthesis.

This equipment will be used to carry out an approved experiment to probe the nuclear equation of state (EOS) by measuring triple-differential cross sections for neutrons from high-multiplicity collisions of equal-mass nuclei as a function of mass number and bombarding energy. Calculations indicate that this experiment can probe the EOS with good sensitivity, and measurement of collective flow of neutrons will provide information complementary to Plastic Ball and Streamer Chamber measurements of charged particles. The wide dynamic range of neutron energies from about 20 MeV to nearly three times the kinetic energy per nucleon of the projectile, is in marked contrast to the Plastic Ball measurements with energies from about 40 to 200 MeV/nucleon. The ability to detect neutrons over this wide dynamic range of energies (and in many detectors simultaneously) compensates for the reduced neutron detection efficiency.

This is a proposal by the Medium-Energy Nuclear Physics Group at Kent State University (KSU) to study Gamow-Teller strength, stretched-state excitations, analyzing powers, and polarization-transfer observables in (p,n) reactions supplemented by (n,p) reactions; to probe the nuclear equation-of-state (EOS) by measuring triple-differential cross sections for neutrons from high-multiplicity collisions of equal-mass nuclei as a function of mass number and bombarding energy, where the azimuthal angle of the reaction plane is determined by measuring the transverse velocities of charged fragments emitted in a collision; and to probe nucleon and nuclear structure via electron-induced reactions. KSU is committed to an experiment to determine the electric form factor of the neutron by using the KSU polarimeter to measure the polarization of the recoil neutron after quasielastic scattering of a longitudinally-polarized electron from an unpolarized neutron in deuterium. Also KSU has submitted letters of intent to CEBAF for experiments on the charge form factor of the neutron, the photoproduction and electroproduction of rho mesons, neutron knockout in the quasielastic (e,e'n) coincidence reaction, and two-nucleon knockout (e,e'2N) reactions. The group is continuing to develop improved instrumentation for the detection of neutrons. Current projects include improved neutron polarimeters and higher efficiency neutron detectors.

This grant provides a state-of-the-art MicroVAX 3500 computer system for use by the Kent State University Intermediate Energy Nuclear Physics faculty and students involved in NSF sponsored research. Essential features of the system include: fast processing time for large scale data replay, line-shape fitting, and analysis codes; large memory; high speed communications; and multi-user environment.

Protein kinases are ubiquitous enzymes that qare frequently involved in signal transduction processes. The major focus of the proposed work is to elucidate the roles that protein kinases may play in photoregulated leaf development in plants. A suite of partial cDNA clones from the garden pea have been sequenced and found to encode protein serine/threonine kinase homologs. Messenger RNA levels corresponding to the five cDNA clones accumulate differentially when pea seedlings are exposed to white light. Experiments are proposed to understand the photoregulated expression of these putative kinase genes at the RNA and protein level. Full-length cDNA clones will be isolated and sequenced in order to understand more fully the structure of the putative kinases. The photoreceptor system(s) controlling mRNA accumulation and gene transcription will be determined. The polypeptides encoded by the pea cDNA clones will be isolated after expression in bacteria and used to generate antibodies. The antibodies will be used to study the accumulation, distribution, and biochemical properties of the protein kinases. Transgenic tobacco plants expressing the pea cDNAs will be produced in order to understand the potential role these enzymes play in the transduction of light signals.

Mevalonic acid (MVA) is the unique precursor for metabolically essential compounds terms "isopentenoids" (e.g., ubiquinones, dolichols, cytochromse a3, juvenile hormones, prenylated proteins, etc.). Since many isopentenoids cannot be obtained from dietary sources, continuous MVA synthesis is required for life. The regulation of MVA synthesis at the molecular level is poorly understood in eukaryotes in general and insects specifically. Moreover, insects cannot synthesize the sterol nucleus, therefore, in contrast to sterologenic organisms, their regulation of MVA synthesis is signaled exclusively by nonsterol isopentenoid availability. Thus, it is possible that a firm understanding of insect nonsterol isopentenoid-mediated regulation of MVA synthesis might provide unique insight into this process in other eukaryotes. We propose to use immortalized Dipteran Kc and secondarily Aedes albopictus C7-10 and Aedes aegypti ATCC-125 cells) as models to define nonsterol-mediated regulation of MVA synthesis. 3-Hydroxy-3-methylglutaryl coenzymeA reductase (HMGR) is the enzyme which catalyzes MVA synthesis from HMG-CoA. In addition, Drosophila Kc cell HMGR Vmax activities are congruent with absolute in vivo MVA synthesis rater. Therefore, an understanding of nonsterol isopentenoid-mediated control of Kc cell HMGR metabolism should also define the regulation of MVA synthesis. Molecular probes for Drosophila HMGR protein and mRNA will be used in conert with detailed radio-metabolic flux analyses to delineate Kc cell nonsterol-mediated regulation of MVA synthesis. Three hypotheses will be addressed: (1) nonsterol isopentenoid-mediated regulation of immortalized Dipteran cellular HMGR metabolism is primarily posttranslational. (2) C15, C20-polyprenols and/or C15, C20-polyprenl-1-pyrophosphates signal MVA-mediated down regulation of Kc cell HMGR, and (3) regulatory isopentenoids modulate Kc cell HMGR function by altering the phosphorylation status of HMGR and/or ancillary proteins. This effort will be facilitated by the use of both intact and perforated Kc cells. The proposed studies will generate new fundamental information about nonsterol isopentenoid-mediated regulation of Kc cell MVA synthesis/HMGR metabolism. Lastly, MVA-derived products are required for insect development, oogenesis, and othe ress Therefore, an understanding of different Dipterans' MVA metabolism and regulation might reveal species specific targets for new chemical/biological insecticides.

9409265 Anderson Research will be carried out in nucleon and nuclear structure via electron and proton induced reactions. Polarization transfer in neutron-proton charge exchange reactions will be used to probe the medium effects on the nucleon-nucleon potential. The charge- structure of the neutron will be probed in polarization transfer studies of electron-induced neutron knockout reactions. A high- efficiency neutron polarimeter will be assembled and calibrated for these purposes. The charge exchange reactions will be carried out at the Indiana University Cyclotron Facility, while measurements of the neutron's charge structure will be carried out at the MIT/Bates lab and at CEBAF. Other related experiments will also be carried out at these laboratories. ***

This three-year grant will support the research of three faculty members, two post-doctoral fellows, and three graduate students to perform a variety of experiments in medium-energy nuclear physics. Much of the activity of the group is focused around measurements of the neutron polarization. These experiments include studies of the complete set of spin observables in the reaction of polarized protons with a variety of targets at the neutron polarization facility at the Indiana University Cyclotron Facility. These experiments use specialized detectors developed by Kent State to measure normal and sideways polarizations of the neutron simultaneously. Measurements with 2H 3He, and 4He targets would study the nucleon-nucleon interaction part that is mediated by a single pion or rho meson. Other experiments would study the isovector response of the nuclear continuum and the isovector giant dipole resonance. The determination of the electric form factor of the neutron would be obtained from neutron polarization measurements using polarized electron beams at the MIT-Bates Laboratory and the Thomas Jefferson National Accelerator Facility (TJNAF). Other experiments include a search for three-body forces and a search for pion-field effects. This grant would also support experiments with the Crystal Ball at the Brookhaven National Laboratory AGS and the CLAS detector at Hall B at TJNAF in order to study neutral baryon resonances and nucleon resonances, respectively.

9714594 Schlesinger The objective of this research is to demonstrate the use of partial reduction as a means of removing the iron from "red mud", produced in the purification of aluminum using the Bayer process. Red mud contains high ferric-oxide, resulting in its name, as well as significant percentages of alumina, soda, silica, lime and titania, and might prove a valuable source of these materials. Currently red mud is disposed of at sea or in shallow lagoons along ocean shores or river banks. Because of the less-hazardous nature of red mud, little research attention has been given to reducing the volume of disposed material or the recovery of its constituents. Several characteristics of red mud make the development of an economically viable treatment process difficult, e.g. the moisture content, the extremely fine particle size and the refractory nature of most of its constituents. One of the most challenging aspects lies in finding a way of separating the iron it contains (in the form of hematite). Several techniques have been attempted, but they have been unsuccessful because of their high energy requirements and because they produce a slag which is difficult to process further. This research is aimed at reducing the hematite to magetite or wustite, which are ferromagnetic and can be recovered by low-gradient magnetic separation processes. This would require lower furnace temperatures, reducing energy costs and minimizing interaction among the other constituents in the mud. The research program will focus on bench-scale reduction experiments varying the type of mud, reduction temperature and the choice and amount of reducing agent; quantitative process mineralogy techniques will be used to assess the degree of reduction achieved and to generate a characterization profile of U.S. red muds. A second focus involves demonstrating the use of low-gradient magnetic separation in recovering iron from the partially reduced materials. Recent advances in magnetic separation have shown promise in iron recovery from other fine-grained process wastes and their integration into a red mud treatment process will help create a viable alternative to present disposal and recovery techniques.
***

9877094

Abstract

This project involves the acquisition of a phosphoimager system for molecular biology research and training. The instrument will be housed in the Department of Biology at Indiana University-Purdue University at Indianapolis and will serve 5 major and two minor users in the department of Biology and two major users in the Department of Chemistry. All major and minor users employ conventional autoradiography, fluorography and/or chemiluminescent detection with film on a routine basis. However, these techniques are limited by the narrow linear response range of film and by the lengthy exposure times required for the fluorographic detection of weak energy isotopes such as 3H and 14C. Phosphoimager analysis has several key advantages over autoradiography and fluorography. First, phosphoimager screens have a linear response range of five orders of magnitude as opposed to only two for autoradiography. Second, phosphoimager exposure times are generally only one tenth those required for autoradiography and fluorography. This is especially advantageous for the detection of weak energy isotopes where fluorography exposure times of weeks or months are often required. Third, the phosphoimager data can be quantified and imported into computer drawing programs for accurate quantitative analysis. This system will complement existing molecular biology equipment in the laboratories of the major and minor users. Moreover, the high degree of accuracy and efficiency afforded by phosphoimager analysis will improve the quality and quantity of molecular biology research and teaching performed by the investigators.

0072240
Anderson
This project is centered around the study of the structure of the
nucleon, primarily by the measurement of the electric form factor of the neutron. This group will play a pivotal role in an approved experiment at the Thomas Jefferson National Accelerator Facility (JLab) designed to measure the electric form factor of the neutron as a function of momentum transfer. Because the neutron is overall neutral, the electric form factor is very sensitive to the details of the constituents and their motions. This measurement provides one of the best experimental tests available for any model of the nucleon and is generally considered to be one of the most important measurements to be performed in intermediate-energy nuclear physics. The experiment will be performed by the scattering of longitudinally polarized electrons from a liquid deuterium target and detecting the knock-out neutron in coincidence with the scattered electron. The Kent State group has the responsibility for providing the neutron detector array for this experiment. The array consists of thirty-six separate neutron detectors arranged so as to both
detect the neutron and determine its spin. The transfer of the spin from the electron beam to the neutron is directly related to the charge form factor of the neutron. The detector array is called a neutron
polarimeter. The Kent State group has used a similar polarimeter in
experiments at other accelerator facilities and will have the
responsibility for the installation, operation, and maintenance of this
detector array for the experiment. This project will be part of the
Ph.D. dissertation research of two Kent State graduate students.
Support from this award will also enable this group to finish the
analysis of experiments performed at the Indiana University Cyclotron
Facility to study the spin dependence of the nuclear force, to search
for three-body contributions to the nuclear force, and also to analyze
measurements of baryon resonances using the Crystal Ball Spectrometer at the Brookhaven AGS accelerator. These measurements were performed with earlier support from the National Science Foundation and are part of the dissertation projects of three Kent State graduate students.

There are two main research areas that will be explored with this research grant. Both of these areas will provide training and dissertation research data for Ph.D. students from Kent State. First, the group will be investigating the structure of the nucleon, i.e., of the neutron and proton. The primary project here is to study the distribution of electrical charge inside the neutron. Although the neutron has no net electrical charge, the three quarks that make up the neutron have electrical charge: plus charge for the "up" quark, and minus charge for the two "down" quarks, with the charges of all three summing up to zero. Since the up quark tends to be closer to the center of the neutron, and the down quarks more on the surface, there is a distribution of charge within the neutron, with more plus charge near the center, and more minus charge near the surface. Precision measurements of this charge distribution will be made at Thomas Jefferson Laboratory, using equipment developed at Kent State. These measurements are of considerable scientific importance, because they provide constraints on theoretical models attempting to describe the quark structure of the neutron. The second project is to study the origin of the intrinsic angular momentum, known as the spin, of the proton. This project has been underway for several years with the STAR detector system at the Relativistic Heavy Ion Collider (RHIC) at Brookhaven National Laboratory; although the total spin of the proton is known, its origin in terms of the interactions and motions of the quarks that makeup the proton is not well understood. Measurements performed with the RHIC accelerator are now providing a clearer picture of the structure of the proton.

The second main area of research is the study of the "correlated pair" structure of nuclei. In the dominant model of nuclear structure, the neutrons and protons move independently of each other in stable quantum orbits inside the nucleus, much like the orbits of the electrons surrounding the nucleus. The developers of this model, called the nuclear shell model, shared the Nobel Prize in 1963. Recent research at Brookhaven National Laboratory and Thomas Jefferson Laboratory has shown that about 20% of the time, a given neutron or proton, instead of moving in an independent orbit, will be part of a pair of particles moving back-to-back at high velocity. These are called "correlated pairs" because their velocities are similar in size, but back-to-back. A fascinating result from that earlier research is that neutron-proton pairs occur 20 times more often than neutron-neutron pairs or proton-proton pairs. This finding has important implications for understanding neutron stars. These results came from the study of the carbon nucleus. The group will be extending that work to the helium nucleus, and observing correlated pairs for a larger range of velocities than were explored in previous work.

The broader impacts of this research include an increased understanding of the physical universe distributed through refereed journals of nuclear physics and the training of doctoral-level physicists needed in industry, medical physics, and academia.

This Collaborative is between the University of California, San Francisco Cancer Center (UCSFCC) and San Francisco State University (SFSU). It will initiate two pilot Research Projects and two Planning and Development activities for collaborative undergraduate and/or pre- doctoral research training in cancer biology. The collaborative pilot Research Projects will involve two teams of SFSU/UCSFCC investigators; both collaborations will consist of a newly hired SFSU. Assistant Professor of Biology and a senior UCSF faculty member who is also a member of the UCSFCC. UCSF Professor Elizabeth Blackburn, Ph.D. will serve as mentor and co-investigator with Sally Pasion, Ph.D. to characterize telomere ends in fission yeast DNA replication and repair mutant. Wilfred Denetclaw, Ph.D. (appointment as a SFSU Assistant Professor of Biology effective September 2000) will be mentored by his former UCSF post-doctoral supervisor Professor Charles Ordahl. Their collaborative Research Project will focus on the biological markers for dermomyotomal myotome progenitor cells and their epithelial-to-mesenchymal transition into the myotome layer. The research questions and mentoring commitments built into each collaborative pilot Research project are designed to result in the submission of RO1 proposals during the third year of this P-20 planning grant. Student research training will be the focus for the Planning and Development section of this proposal. One pilot Planning and Development activity will use a joint SFSU and UCSFCC faculty committee to develop a more aggressive and concerted placement process/program to recruit SFSU undergraduate (MARC and MBRS- RISE) and SFSU MA/MS degree (NIH Bridge and MBRS-RISE) students into UCSFCC laboratories specifically will review and evaluate the past involvement of, 1) SFSU undergraduates (MARC and MBRS- RISE) in UCSF summer research experiences and 2) the academic year thesis research experiences of SFSU RISE) in UCSF summer research experiences and 2) the academic year thesis research experiences of SFSU MA/MS degree students whose research was executed in UCSF faculty laboratories as a basis to develop an action plan that will provide an excellent training experience in an environment permeated with a cancer biology focus. The second pilot Planning and Development activity will be directed at doing the ground work necessary to develop a joint R-25E or R-25T or T- 32 pre-doctoral training proposal during the third year of this P-20 project. Realization of this goal will require serious planning through joint UCSFCC-SFSU faculty meetings and retreats to 1) identify and prioritize short and long-term pre-doctoral student cancer research training goals and 2) develop a mutually acceptable collaborative pre- doctoral cancer research training program within the guidelines of a R- 25E or R-25T or T-32 funding mechanism. A core element of any final proposal will be the application of recruitment and retention strategies that will result in an increased enrollment of under-represented strategies that will result in an increased enrollment of under-represented minorities into and graduation from Ph.D. programs.

Protein kinases are regulatory enzymes found in all cell types. They are involved in a vast array of responses to hormonal, nutritional, developmental, and environmental stimuli. The overall goal of the work proposed is to elucidate the roles played by selected protein kinases in the development of young seedlings. Two protein kinases from Arabidopsis named WAG1 and WAG2 are the focus of this proposal. Mutations in both WAG1 and WAG2 abolish the expression of these genes. Nevertheless, the wag1 and wag2 single mutants exhibit no detectable affect on the plant. However, when the wag single mutants are genetically crossed to generate wag1/wag2 double mutants, they do show a readily observable difference compared to their non-mutant counterparts. Seedlings of the double mutants exhibit a pronounced root growth defect when grown on vertically-oriented Petri plates: the roots possess a wavy pattern. Root waving of this type is observed in normal seedlings only when the Petri plates are leaned backward from vertical (inclined at pitches less than 90). It is proposed that WAG1 and WAG2 are negative regulators of root waving in Arabidopsis.

To elucidate the roles that WAG1 and WAG2 play in root growth, the specific aims of the proposed research are:

(1) To ask whether altering the amount or enzymatic activity of WAG1 and WAG2 affects the root waving response in transgenic Arabidopsis seedlings.

(2) To explore the underlying mechanisms for the wavy root phenotype in wag1/wag2 mutants, particularly with regard to the effect of gravity on root growth and the transport of and response to the plant growth hormone auxin.


Broader Impacts:
This project will provide training opportunities for an undergraduate student, a graduate student, and a postdoctoral associate. The two WAG genes under study encode protein kinases that when mutated confer an unusual growth pattern on seedling roots. Understanding the role of the WAG kinases will provide fundamental insight into the cellular pathways regulating root development that have not yet been defined. Root waving can be viewed as an adaptive response that may enhance the ability of a root to navigate its way around impenetrable objects encountered in the soil. As such, understanding the process of root waving and the genes that regulate it might contribute to other research areas, such as ecological physiology. Moreover, manipulating root waving genetically might improve a young seedlings ability to become established in the field, and thus provide agricultural benefits as well.