Randolph V. Lewis - US grants

peptides; neuroendocrine system; gene expression; biological information processing; physiologic stressor; nucleic acid sequence; stress; small cell lung cancer; chemical cleavage; adrenal medulla; genetic manipulation; messenger RNA; tissue /cell culture; radioimmunoassay; monoclonal antibody; radioassay; chromatography;

The ultimate goal of this project is to understand the processing of proenkephalin and the regulation of that processing. Recently the enkephalin precursor in adrenal medulla has been identified and sequenced via DNA cloning. From this sequence and the sequences of enkephalin-containing polypeptides (ECP's) we have isolated, it is clear that the post-translational processing of proenkephalin requires a trypsin-cleavage like enzyme. Using chromaffin cells in culture we will use a pulse chase to determine the order of these cleavages. We have identified an enzyme(s) in adrenal extracts, which has a pH optimum consistent with the low pH within chromaffin granules and which has been shown to cleave at proenkephalin cleavage sites using peptide substrates. We propose to purify this enzyme and study its physiochemical properties, its relationship to other proteases, and whether it is present in other enkephalin-producing tissues such as gut and brain. This enzyme also appears to be regulated in some manner as all possible cleavage sites are not cleaved normally but rupture of the granules and dilution in buffer results in much greater cleavage. How this regulation occurs will be determined. In addition to the regulation of this enzyme we have evidence suggesting that epinephrine containing granules have a greater proportion of enkephalins and ECP's than do nonepinephrine granules. Whether this is due to regulation of the trypsin-like enzyme or regulation of proenkephalin synthesis will be determined using chromaffin cell primary cultures. The overall result will be an understanding of how the post-translational processing of proenkephalin occurs and how it is regulated to result in the normally produced ECP's in adrenal tissue and enkephalins in neural tissue.

The ultimate goal of this project is to understand the processing of proenkephalin and the regulation of that processing. Recently the enkephalin precursor in adrenal medulla has been identified and sequenced via DNA cloning. From this sequence and the sequences of enkephalin-containing polypeptides (ECP's) we have isolated, it is clear that the post-translational processing of proenkephalin requires a trypsin-cleavage like enzyme. Using chromaffin cells in culture we will use a pulse chase to determine the order of these cleavages. We have identified an enzyme(s) in adrenal extracts, which has a pH optimum consistent with the low pH within chromaffin granules and which has been shown to cleave at proenkephalin cleavage sites using peptide substrates. We propose to purify this enzyme and study its physiochemical properties, its relationship to other proteases, and whether it is present in other enkephalin-producing tissues such as gut and brain. This enzyme also appears to be regulated in some manner as all possible cleavage sites are not cleaved normally but rupture of the granules and dilution in buffer results in much greater cleavage. How this regulation occurs will be determined. In addition to the regulation of this enzyme we have evidence suggesting that epinephrine containing granules have a greater proportion of enkephalins and ECP's than do nonepinephrine granules. Whether this is due to regulation of the trypsin-like enzyme or regulation of proenkephalin synthesis will be determined using chromaffin cell primary cultures. The overall result will be an understanding of how the post-translational processing of proenkephalin occurs and how it is regulated to result in the normally produced ECP's in adrenal tissue and enkephalins in neural tissue.

The Microchemical facility at the University of Wyoming has been operating for three years now. Services available currently include: 1) protein-peptide sequencing, 2) amino acid analyses both hydrolysis and physiologic fluid, 3) DNA synthesis, and 4) peptide synthesis. The sequencing and amino acid analysis services have been used by twelve on-campus investigators in four departments and by twenty off-campus investigators from numerous other institutions. A critical factor in the efficiency and productivity of this laboratory has been the skill and dedication of the two operators. However, it is clear that their capacity has been reached and the solution to the continued optimal operation of this facility is to update the sequencers to include the automated on-line PTH analyzes and to have an automated, flexible chemistry amino acid analyzer. Those are the items requested in this proposal. There are four major users and a large number of minor users both on- and off-campus.

It has recently been demonstrated that the transparency of the mammalian lens is apparently due to short range order among the major proteins of the lens, the crystallins. An understanding of the nature of this order is an essential step in obtaining a molecular description of cataract formation (in which this order is disrupted). The major objective of this research is to describe intermolecular interactions between bovine lens crystallins in the form of a two dimensional spatial map of binary protein proximity relationships. This map will be generated by a systematic series of measurements employing three crystallin fractions. The potential existence of crystallin/crystallin interactions will be addressed within both homogeneous crystallin solutions and in all possible binary mixtures of crystallins as a function of protein concentration. The functional significance of any interactions detected will be evaluated based on the observation of the onset of increasing optical transparency seen at high protein concentrations in lens protein extracts. The selection of the experimental methods to be used is based on a desire to minimize perturbation of protein surfaces and a need to make measurements over an unusually wide range of protein concentrations. On this basis, the techniques of dynamic light scattering, chemical crosslinking and fluorescence energy transfer have been chosen to examine crystallin/crystallin interactions. The effect of calcium and sodium chloride will also be explored because of the known effects of these agents on the aggregation of crystallins. The resulting supra-molecular description of the lens in terms of the relative topological locations of the crystallins will then serve as a future framework within which to study cataract related phenomena.

9553642 Lewis The Annual NSF EPSCoR Conference provides a forum for the exchange of information between the National Science Foundation (NSF), members of the state EPSCoR programs and many other parties with interests in the EPSCoR program. During the Conference, progress and evaluation are discussed as well as opportunities and strategies for the coming year. The State of Wyoming proposes to host the 1995 Conference in Jackson Hole, Wyoming on September 29-29, 1995. The theme of the Conference would be "EPSCoR: Design of a Partnership for Success". The conference will focus on issues that are important for achieving the goals foe EPSCoR. Topics for discussion will include: 1) Assessment and evaluation methods at the state and national levels; 2) Individual state programs and methods for success; 3) Opportunities for collaboration between states and federal agencies to further advance EPSCoR goals; 4) Private sector involvement in EPSCoR programs; and 5) Federal policy changes and their effects on state EPSCoR programs. These objectives will be met through a combination of invited speakers, symposia, poster sessions and displays, and adequate time for discussion. Attendance at the meeting is expected to include, at least the following groups of people: Project Directors and Administrative Assistants, State EPSCoR Committee members, University Administrators, State Officials of Higher Education, EPSCoR scientists, State Legislators, Private Sector members, and representatives from other Federal Agencies and Programs.

minerals; biotechnology; biomedical resource; bioengineering /biomedical engineering; biomaterials; Invertebrata;

9806999
Lewis
The long-term goal of this proposed research is to develop strategies for the design of biomimetic major ampullate silk by uncovering the structure-function relationships of naturally ocurring spider silk proteins and their materials properties. This will be accomplished through two major objectives. First, the nucleotide and amino acid sequence variation in major ampullate silk genes among divergent spider species will be characterized. The second objective is to study the structure of the proteins in the solid fiber to understand what provides the elastic and strength properties. These data will provide the basis for construction of second generation spider silk biomaterials with properties designed into them.

Natural materials have been a part of our culture for centuries but only in the past few years has the study of these materials reached the molecular level. This deeper understanding of biological materials has led to the new field of biomimetics. One of the materials which has generated considerable interest is spider silk. Spider silk fibers possess a numbrr of unique properties when compared to other natural or manmade fibers. Almost all the applied research on spider silk has focused on major ampullate silk and its military and industrial potential. This material has a tensile strength in excess of 300,000 psi and an extension approaching 35%. This two factors make it one of the toughest materials ever tested. These unique properties suggest promising applications in fibers, films and composite materials. There is no question that the development of spider silk as a biomaterial will have a substantial impact on society. These new materials would provide a number of new products as well as improvements on current ones. These products will be fibers, films and composites with spider silk as the key component. The investigators have taken some of the necessary steps but the information generated by this proposal will substantially enhance the ability to produce major ampullate silk as a biometerial as well as be the basis for developing synthetic silk analogs with designed mechanical properties.

This project will enhance the research infrastructure at the University of Wyoming, the state's only university and its only four-year institution of higher education. Wyoming chose to focus the improvement of its research activities around the theme of science and engineering in harsh environments, a theme appropriate for the state considering its temperature extremes, limited precipitation, high sunlight impact, saline soil and population impact in limited geographic areas. The specific scientific areas selected to be focused on 1) Natural Resources Sciences, and 2) Materials Sciences.

The University will hire at least 10 new faculty members (6 for Natural Resources and 4 for Materials). These new hires are expected to engage in and develop multidisciplinary research teams that can improve the research competitiveness of the University and have a potential economic impact on the state. Multi-use research equipment will be acquired to assist both new and existing university researchers. A faculty development program will be initiated to assist the research competitiveness of junior faculty. Programs will be supported that assist in the development of research-oriented small businesses within Wyoming, that involve high school and undergraduate students in research activities, and that expose K-12 teachers to university science and mathematics instruction.

Improvement of the University of Wyoming's research infrastructure is important not only to the ability of the University's researchers to successfully compete for federal research funds but also to the development of the state as a whole. Wyoming's economy has been historically based on natural resource use. Support of research infrastructure improvements at the University of Wyoming can play a significant role in the diversification of the state's economy by assisting in the development of technology-related businesses in the state.

With this award from the Chemical Research Instrumentation and Facilities (CRIF) Program, the Department of Chemistry at the University of Wyoming will upgrade a 400 MHz Solid State Nuclear Magnetic Resonance Spectrometer. This equipment will enable researchers to carry out studies on a) zeolite catalyzed reaction chemistry; b) amorphous materials characterization and polyamorphic dynamics; c) spider silk and other biopolymer elucidation; d) ion diffusion in fuel cell and battery materials; e) solid state luminescence matrix characterization; and f) optical pumping and detection enhanced spectroscopy.

Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful tool available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, to characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solution. Access to state-of-the-art NMR spectrometry is essential to chemists who are carrying out frontier research. The results from these NMR studies will have an impact in a number of areas including materials chemistry, battery research, and catalysis.

Certain orb-weaving spiders are known to recycle their webs, some as often as every day. They accomplish this despite the fact that some web fibers, namely major and minor ampullate silk, are highly resistant to common proteases. Spider proteases that degrade these solid fibers are therefore expected to have novel properties. Using digestive fluid from the orb-weaving spider Nephila clavipes, major ampullate silk-degrading or "silkase" activity was identified. The objective of this research is to determine protein sequences that distinguish peptidase isoforms with and without major ampullate silk-degrading activity. By examining enzymatic and sequence data from several spider species, structural and functional features will be correlated. Enzymatic activities of recombinant and native peptidases will be compared using synthetic or soluble silk fibroins or native silk fibers. To test the hypothesis that certain peptidase sequences are key determinants for the presence of major ampullate silkase activity, altered recombinant peptidases will be analyzed.

Broader Impact: Characterization of a silkase with novel enzymatic features will advance our understanding of enzymes that degrade protease-resistant solid fibers and could lead to new biotechnological applications. Students involved in this research will benefit greatly from the collaboration between research groups interested in protein structure/function relationships from the perspectives of the silk fibers and fiber-degrading peptidases. The research will also provide a rich environment for hosting high school students from underrepresented groups in a summer Student Research Apprenticeship Program and undergraduates receiving NSF-EPSCOR summer research awards.

A workshop on management strategies and future planning for the NSF EPSCoR program will be held in June
2003. Topics to be addressed include the following: strategies for building effective state committees, the role of EPSCoR in economic development, human resource development at research universities, effective evaluation strategies, effective communication with the public, and prioritizing areas for EPSCoR investment. The resulting documnetation will be a resource manual and a set of recommendations for future strategies.

ABSTRACT:

Spiders have been using protein-based nano-materials with the ability to self-assemble into fibers for over 450 million years. However, it is only in the past 10 years that an understanding of the basis for this process has emerged. Investigators from the University of Wyoming, Nexia Biotechnologies Inc., the University of Bologna, and the University of Kansas will work together in testing three basic hypotheses and engineering concepts. (1) Amino acid sequence motifs from spider silk can be used to create nano-springs and self-assembling elastic materials. (2) The elasticity of the individual molecules and the materials will be proportional to the number of elastic motifs they contain. (3) The elastic modulus of the materials can be varied by using different amino acid sequence motifs found in various spider silks.

Possible applications of the spider silk proteins seem widespread. The molecules themselves can serve as springs in a variety of nano-materials based applications. If our hypotheses are correct we can control the elasticity and elastic modulus of each protein spring. Since these nano-materials will self-assemble into fibers or films they can be used in any application where the unique materials properties of these silks will be of advantage. Uses range from artificial ligaments and tendons to protective clothing to composite materials.

The goal of this research is to understand the mechanical properties of tubuliform silk and its evolutionary origin. Tubuliform silk, which is used to form the egg case of orb-weaving spider, is unique in several important ways. First, its sequence must be very different from the other orb-weaving spider silks in that it contains a very different amino acid composition. Second, it is produced only for a short period in the spider's life, just before the eggs are laid. Finally, its mechanical properties show a high tensile strength but this is combined with a low ability to withstand bending without fracture. The work plan is: 1) Four species of spiders from a broad section of the orb-weaving, derived orb-weaving and cob weaving spiders will be used. 2) The tubuliform silk fibers will be collected for mechanical and biophysical testing. 3) Genomic DNA libraries and cDNA libraries will be constructed, these libraries screened, and positive clones sequenced. 4) Comparisons will be made between these silk proteins and other silk proteins to help determine the role the protein sequence plays in mechanical properties and the evolutionary path of this silk.

The broader impacts of this research involve both the general public and students. Spider silks represent a unique answer to the production of biomaterials for applications ranging from protective clothing to medical products to composite materials. On average 3-4 undergraduates will work in the laboratory every semester and 2-3 high school minority students every summer. At least10 presentations are given each year on spiders and spider silk to elementary and high school groups and service clubs in the state. Information, pictures and video clips are provide each year for several publications and TV and radio stations including PBS, National Geographic, CBS, NBC, etc as well as a number from outside this country, BBC and CBC.

DESCRIPTION (provided by applicant): Spider silk, which has been evolving for over 450 million years, has a tensile strength greater than steel and elasticity greater than nylon. A number of spider silk genes have been cloned and sequenced revealing specific amino acid motifs that have been conserved for over 125 million years. The key element in taking the next step toward generating bio-based materials from spider silks will be to move from the current descriptive data to predictive knowledge. These experiments will provide the predictive knowledge enabling the design of materials with very specific elastic and strength properties for each different medical application. This renewal is designed to continue testing two basic hypotheses and engineering concepts. 1) The elasticity of the materials will be proportional to the number of elastic motifs. 2) Varying the sequence of the elastic regions will vary elastic (Young's) modulus. A brief workplan is described here. 1) Continue the expression and purification of the proteins. 2) Optimize the spinning and film making process to maximize desired materials properties. 3) Test mechanical properties of films and fibers. 4) Determine the structure of the protein in films and fibers by FTIR, CD and solid state NMR. 5) The elasticity and tensile strength data will be correlated with the number and sequence of each type of motif to produce a prediction algorithm for elastic and tensile strength properties. 6) Based on 5) new genes will be constructed to match the properties needed for ligament and tendons. The past two years of work have produced a number of new genes, purified proteins and fibers from those proteins. Improvements in fiber spinning and testing of those fibers is currently in progress. This project is highly significant for several reasons. First it will provide a basic understanding of elasticity and tensile strength in spider silk proteins. Specifically, it will reveal what controls the amount of elasticity and elastic modulus and if these two factors can be varied in a predictable way. Second this project will advance our ability to use spider silk as a biomaterial. If our hypotheses are correct we will learn how to control the elasticity and other materials properties by controlling the protein sequence. Possible applications of spider silk range from artificial ligaments and tendons to bandages for burns to composite materials for multiple applications.

ABSTRACT NOT PROVIDED

Proposal: EPS-0447681

Proposal Title: Wyoming NSF EPSCoR Research Infrastructure
Improvement

Institution: University of Wyoming

The Wyoming EPSCoR Research Infrastructure Improvement award will build capacity and capability for national competitiveness in Natural Resource Sciences, especially emphasizing ecological topology of different spatial and time scales as it relates to ecosystem and global change: Currently, the university has 20 faculty members in diverse departments for this very broad, interdisciplinary area. The award will provide partial start-up support for five new hires to fill specific needed niches in a newly initiated interdisciplinary PhD program. Equipment and committed technical staff will be supported to enhance the Stable Isotope, Nucleic Acid Exploration, and GIS facilities, thereby strengthening research competitiveness and fostering additional collaborations in this research focus area and contributing to the development of a critical mass of research and educational expertise necessary for large, multi-investigator, competitive research programs. This focus relating to ecosystem responses to global change has significant current merit and importance both for Wyoming and, more broadly, for national and international concerns. There is growing recognition of the need for modeling and understanding ecological processes at different spatial scales. This project will provide a research and education focus related to that need for the university. More importantly, Wyoming's strength in geological studies will give added value in being able to address questions in different time scales as well as spatial scales. This perspective will be of particular value in informing policy decisions.

Integration of research and education will be emphasized in all aspects of the project. In the faculty recruitments researchers who are also excellent teachers will be sought to strengthen the new interdisciplinary PhD program. The award will supplement the university's level of graduate student stipends and this greater level of support will require that participating students take a course in Teaching for Scientists and Engineers. A different kind of graduate student support is to be provided for graduate student mentors for the Science Education program. Students receiving this mentorship support will assist secondary science education students in a summer research experience. Undergraduate fellowships for university and community college students will engage these students in research experiences. Outreach efforts will expand the number of high students participating in a previously successful summer research program. In addition, technical assistance will be engaged for efforts to increase public awareness of the role of research in higher education and its contribution to the state's economic growth and especially to make high school students and their parents more aware of undergraduate research opportunities.

With support from the Major Research Instrumentation (MRI) Acquisition Program, the Department of Chemistry at the University of Wyoming will acquire a single crystal X-ray diffractometer. The diffractometer will be used in the structural characterization of inorganic and macromolecular enzyme models, fluoroalkylphosphine complexes that are potential hydrogenation catalysts, and linear oligometallic complexes for photophysics studies. Other uses are in the areas of silk fiber structure analysis, carbohydrate and natural product synthesis, surfactant synthesis, and polypeptide folding dynamics.

The X-ray diffractometer allows the determination of accurate and precise bond distances and angles between atoms in a molecule. It essentially maps out the three dimensional structure of a molecule, and the spatial arrangement of the molecule relative to the neighboring molecules. The composition of a solid material is fully described by analyzing the diffraction pattern. This equipment will allow the University of Wyoming to better train students across the chemical sciences at both the undergraduate and graduate levels with a modern diffractometer system and software.

This proposal describes a Track 1 project developed by the University of Wyoming in partnership with several schools with high Native American and Hispanic communities.
The goals of this project are to motivate and prepare students for self-directed learning and to nurture an appreciation for discovery that will lead to careers in science. The project seeks to engage communities to deepen understanding of contemporary STEM issues. The project will focus on the interdisciplinary nature, unifying concepts and ethical considerations of science from the perspective of "What is Alive?" In order to intervene in the prevalent socio-educational culture that now directs students into non-college curricular tracts in the 9th grade, the program will target 7th and 8th grade classes. Initially, communities with significant Native American and Hispanic communities will been targeted; however, the long-term goal will be to reach all school districts in Wyoming, in part, via distance delivery technologies and continuing education workshops. The broad program goals will be attained by meeting four specific objectives:
* Enhance awareness and understanding of contemporary STEM issues, opportunities and ethics by offering motivational units to students and communities.
* Promote STEM knowledge and skills by delivering interdisciplinary curricular units that align with state standards.
* Advance graduate student learning and outcomes.
* Increase sustainable STEM expertise of 7th and 8th grade science teachers.

Knowledge of the nature and relevance of scientific discovery will provoke sustainable student learning, achievement and career choice.