Frederick Byron Rudolph - US grants

The major goal of this project is to determine how nucleotide metabolism is regulated at both the enzyme and cellular level. Specific studies range from detailed kinetic, physical and regulatory enzyme investigations to the dependence of certain cell types on dietary sources of preformed purine and pyrimidine bases. Three enzymes involved in critical steps in biosynthesis, degradation and interconversion of nucleotides will be studied. The first is adenylosuccinate synthetase which catalyzes the first committed step in AMP formation from IMP. Detailed physical, mechanistic and regulatory investigations will be done. Physical and mechanistic studies will also be done with adenosine deaminase, which catalyzes the irreversible deamination of adenosine or 2'-deoxyadenosine. Site specific mutations will be done in a third study with an unusual form of dihydrofolate reductase, an enzyme involved in deoxynucleotide metabolism. This enzyme which has been isolated from an E. coli plasmid is a small, stable protein which will provide an excellent model system for the study of the role of specific amino acid residues in catalysis and binding. In a related, ongoing study, the requirements of T lymphocytes for a dietary source of preformed purine or pyrimidine bases will be determined. Evaluation of the biochemical basis of this effect will involve analysis of enzyme and metabolite changes as correlated with immune function under different dietary conditions. The combined effect of antimetabolites and diet on the immune system and on lymphoid derived tumors will be determined. These studies should afford new information concerning function and regulation of enzymes involved in nucleotide metabolism and potential new therapeutic protocols for transplant recipients, tumor patients and other patients receiving parenteral or enteral nutritional support.

The objective of this research is to understand, at the enzyme level, the biochemical events which lead to and control solvent production in the butanol/acetone fermentation. Elucidation of the control mechanisms of solvent production will suggest direct butanol selectivity, and yields. The latter determne the economic feasibility of this important fermentation. The activity levels of 12 key product-formation enzymes will be measured at various stages.

Efforts are underway to investigate and develop methodologies for the transformation of butyric-acid clostridia (Clostridium acetobutylicum and related strains) and to investigate the fundamental and applied aspects of their molecular biology and genetics for biocatialysis applications. Little is known about the genetics or regulation of enzymatic activities involved in the formation of primary metabolism products (organic acids and solvents) of strict anaerober such as the butyric-acid clostridia. Mutants deficient in enzymes of solvent forming pathways will be isolated and characterized. These mutant strains will allow the cloning and characterization of genes which complement the deficiencies. This approach will not only allow isolation of the structural genes of the enzymes directly involved, but also enable regulatory elements which may be necessary for expression of these genes to be identified and analyzed.

The objectives of the proposed research are to produce new knowledge on the molecular genetics and enzymology of anaerobic butryic-acid clostridia that will allow beneficial genetic alterations of cell metabolism (metabolic engineering) for a variety of applications. Butryic-acid clostridia have an excellent potential for industrial applications to produce commodity and specialty chemicals. The current knowledge base on the molecular genetics, cell regulation and metabolic engineering of clostridia cells is very narrow, but expanding as a result of current efforts by the Principal Investigators of this proposal. Proposed studies include construction of a variety of cells with altered enzymatic pathways and testing of these cells in batch, continuous and cell-recycle bioreactors to study the stability of these cells and the effectiveness of the recombinant enzymes in product formation.

The objectives of this project are to produce new knowledge on the molecular genetics and enzymology of anaerobic butryic clostridia that will allow beneficial genetic alterations of the cell metabolism for a variety of applications. Butryic acid clostridia have an excellent potential for industrial applications to produce commodity and specialty chemicals. The cur- rent knowledge base on the genetic- s, regulation and metabolism of these cells has been limited, but considerable progress has been made by work on previous grants. This project will continue the study of gene regulation, develop further the expression systems in clostrid- ia, and evaluate other factors in- volved in the regulation of metabo- lic pathways in clostridia.

The goals of the studies described in this proposal are to understand the relationship between molecular structure and functional properties of adenosine deaminase (ADA) and related proteins. ADA is present in all mammalian cells and tissues but the level of the enzyme varies by more than 10,000 fold in a tissue specific manner. ADA has a central role in development of the lymphoid system as genetic absence of the enzyme leads to severe combined immunodeficiency disease and its levels are related to other immunological and neoplastic disease states. ADA function is critical in control of the effects of adenosine in a variety of systems including roles in neurological function, catabolism of nutrients and the vascular system. A number of clinically important purine analogues are used as inhibitors of ADA including 2-deoxycoformycin (pentostatin) and several neurologically active compounds. This enzyme represents an ideal system for detailed analysis as it is available in large amounts in a stable form isolated from cloned genes, can easily be mutated, catalyzes a simple, important reaction and is a relatively small monomeric protein with a zinc cofactor. The crystal structure of the enzyme with a transition state analogue has been determined and a new catalytic mechanism involving the newly discovered zinc atom at the active site has been proposed. The requirement for zinc may be a part of the effect of dietary zinc deficiency on suppression of the immune system. Additional enzymes with similar catalytic function are available to evaluate the general nature of this work. Specific proposed studies include: 1. Identification and functional analysis of specific amino acid residues involved in the catalytic process for ADA. 2. Use of metal substituted ADA to study substrate and inhibitor interactions with the enzyme. 3. Identification and functional analysis of specific amino acid residues involved in the catalytic mechanism of AMP deaminase by comparison of conserved sequences between ADA and AMP deaminase. The results from these studies will provide a clear basis for understanding how ADA functions as a catalyst along with other enzymes with similar mechanisms such as AMP deaminase.

IBN-9507878 Raymon Glantz A broad spectrum of brain activities, from object recognition to postural adjustments, require that neurons and neural circuits make computations. In recent years, neurobiologists have made impressive progress in two related areas: one concerns the biophysical and molecular mechanisms of synapses and the second concerns the mathematical and logical operations which formally describe what whole systems of nerve cells do. In the study of how the visual system works, algorithms successfully model visual tracking and fixation, binocular vision, and motion detection. Future progress in understanding more about the visual system depends upon integrating the biophysical and algorithmic approaches. This will require detailed information about the structure and dynamic properties of neurons involved in particular computations. Many visual systems can compute the direction and velocity of moving targets. Recent studies indicate that directional motion detection is achieved through synaptic operations embedded in the structure of the neuron's receptive field. The same directional mechanisms were independently found in visual neurons in higher vertebrates and in the crayfish. The mechanisms depends upon spatial and temporal asymmetries of the excitation and inhibition within the receptive field. This project focuses on experimental and theoretical studies of the synaptic mechanisms which support directional motion detection. Physiological measurements will quantify the characteristics of the synapses which form the directional mechanism. The simulations will determine if the measured properties of the synapses are sufficient to support the directional mechanism.

The objective of the project is to investigate the metabolic engineering of solventogenic Clostridia using modern molecular biology and genetic techniques. The primary goal of this interdisciplinary proposal is to genetically alter the metabolism of Clostridium acetobutylicum. The proposal describes four areas
of activity; (1) a detailed examination of the regulation of the sol locus genes through a putative repressor, SolR; (2) a molecular analysis of the function of sporOA and of ORF (orfY) gene products, both of which are likely to be important in the expression of the sol locus genes; (3) an analysis of patterns of gene expression using cDNA arrays; and (4) the further development of antisense RNA as a metabolic engineering tool.

Rice University and two University of Houston campuses are joining the Houston Independent School District to create an innovative program that teams master and novice middle school teachers with SMET undergraduate and graduate students, designated as GK-12 Fellows. Seven four-member teams are working together over a fifteen month fellowship period in an exchange of content knowledge and pedagogy.
The program includes the following major features:
* Fellows are introduced to K-12 education by participating in teacher enhancement programs at the participating universities. After spending an entire academic year working with their teacher team-mates, GK-12 Fellows serve as instructors in these same teacher enhancement programs during the last three months of their fellowship.
* Teams design a two-semester project that involves the middle school students of the participating teachers. Projects focus on one of three themes: curriculum development, learning processes, or integration of instructional technology into the K-12 science and mathematics curriculum.
* In addition to their time on K-12 campuses, Fellows participate in a weekly seminar on precollege science and mathematics educational reform.
* Fellows serve as role models for middle school students as they provide instructional assistance to their teacher teammates.
Our universities have a long history of working closely with the local school district to meet identified needs. This program strengthens and expands the already established relationships among the participating institutions, provides K-12 teachers with an exceptional professional development opportunity and their students with enriched instruction in science and mathematics, at the same time preparing graduate and undergraduate students to support K-12 education in their future careers.

This IGERT award supports the establishment of an interdisciplinary research training program in the emerging field of cellular engineering. The program focuses on metabolic and tissue engineering and provides science and engineering students with rigorous educational and research training in the fields of bioengineering, biochemistry, and cell biology. A co-supervision system is created in which students will have an advisor from the Department of Bioengineering and an advisor from the Department of Biochemistry and Cell Biology for guidance of mechanistic and design aspects of research projects. The fundamental curriculum for IGERT trainees includes coverage of scientific ethics, advanced laboratory skills, basic biosciences (biochemistry and cell biology) and engineering systems analysis. Student participants work in cooperative environments including team design projects and an industrial internship program with companies engaged in cellular engineering. This program is coordinated with a pre- and post internship seminar program to maximize the impact of the students' industrial experiences. The establishment of a visiting scientist position and a focused seminar series having different annual themes provides in depth exposure to new areas. A key component of the program will be continued expansion of our successful undergraduate recruitment program for underrepresented minorities to this specialized area of graduate education. Building upon established strengths in interdisciplinary research and education, this training program creates a center of excellence in cellular engineering that will train researchers who can utilize advances in biological sciences to produce innovative and cost- effective biotechnological products in the 21st century.

IGERT is an NSF-wide program intended to meet the challenges of educating Ph.D. scientists and engineers with the multidisciplinary backgrounds and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing new, innovative models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In the fourth year of the program, awards are being made to twenty-two institutions for programs that collectively span all areas of science and engineering supported by NSF. The intellectual foci of this specific award reside in the Directorates for Engineering; Biological Sciences; and Education and Human Resources.