Roger A. Sunde - US grants

This project proposes to examine the metabolism of selenium (Se) leading to Se incorporation into glutathione peroxidase (GSH-Px). GSH-Px is the only known selenoenzyme present in animals, and the Se is present in the enzyme as an amino acid, selenocysteine (Se-Cys), incorporated into the peptide backbone of the enzyme. The mechanism for formation of this Se-containing moiety is unknown. My initial results have indicated that the Se-Cys moiety is formed by the covalent attachment of an inorganic form of Se to an amino acid residue, perhaps serine, already present in the peptide backbone of the enzyme. The major form of the Se in foodstuffs of plant origin is thought to be selenomethionine (Se-Met), but the metabolism of dietary Se-Met to the form used for GSH-Px synthesis has not been characterized. Furthermore, we have shown that the level of dietary methionine can have a dramatic effect on the availability of Se from Se-Met for GSH-Px synthesis, perhaps because Se-Met can be incorporated into protein as a Met analog. Thus the availability of various inorganic and organic forms of Se for GSH-Px synthesis depends on the nature of Se metabolism in the body. To further our understanding of Se metabolism, we intend to pursue the following specific objectives: 1) identification of the origin of the carbon skeleton of the Se-Cys moiety in GSH-Px and thus identification of the mechanism used to form the Se-Cys moiety; 2) utilization of anti-GSH-Px antibodies to determine the role of Se in the regulation of GSH-Px protein synthesis; 3) utilization of the perfused rat liver to further characterize the ability of various Se-containing metabolites to provide Se for GSH-Px synthesis; 4) comparison of the two fates of Se-Met -- Se-Met incorporation into general body proteins versus Se-Met degradation to release Se for GSH-Px synthesis -- to determine Se bioavailability in foods and in "selenized yeast". The overall objective of this research is to better understand Se metabolism and the subsequent availability of Se for GSH-Px synthesis. This knowledge is important because of the widespread use of Se supplementation in animal nutrition, and because of the increasing inclination of humans to supplement themselves with Se.

This application proposes to study the nutritional regulation of selenium- containing enzymes and proteins. Glutathione peroxidase (GPX) is the best characterized selenoenzyme present in animals, and thus this research will have a major focus on GPX. GPX activity levels decrease rapidly during dietary Se deficiency, and we have used anti-GPX antibodies to show that GPX protein levels also fall exponentially and coordinately during progressive Se deficiency. Using a cloned GPX cDNA prove in Northern blot analysis, we have recently shown that the level of Se in the diet regulates the level of GPX mRNA as well as GPX activity and protein, thus suggesting that Se status of animals may regulate Se-containing enzymes such as GPX at the transcriptional level of gene expression. This regulatory mechanism maybe one of the major reasons why the dietary Se requirements for most higher animals, usually determined using GPX as an indicator, are so similar. In the last decade it has become increasingly clear that a number of selenoproteins in addition to GPX are present in the tissues of higher animals, but the nature and function of these selenoproteins are largely unknown. As with other species that are dependent on trace dietary factors for full biological activity, these species should be further characterized. Because activity assays, antibodies or cDNA probes are not generally available for these uncharacterized selenoproteins, our previously developed SDS/PAGE procedure can be used to identify and to quantitate 75Se-labeling of a number of these selenoproteins. This will allow initial studies on these relatively uncharacterized selenoproteins. To further our understanding of Se metabolism, we intend to pursue the following specific objectives: 1) to determine the effect of Se status on the expression of GPX (mRNA, protein and enzyme activity levels) in eukaryotes, and to further characterize this process; 2) to purify and to further characterize one or more of the uncharacterized mammalian selenoproteins. The overall objective of this research is to better understand the nutritional regulation of selenoproteins and selenoenzymes such as GPX. This knowledge may lead to a better understanding of the nutritional biochemistry of Se and it may increase our understanding of selenium's full role in animal and human health.

This proposal requests partial funding for the 1995 FASEB Summer Conference on Micronutrients: Trace Elements to be held June 25-30, 1995 at Copper Mountain, Colorado. An attendance of 130-150 is anticipated based on the following: a) participation an outstanding evaluations of the previous four FASEB conferences; b) continued and expanding interest in both basic and applied problems in trace element research; c) new sessions on hot areas utilizing molecular biology approaches to solve trace element questions; d) the conference site. The conference will focus on recent developments related to the metabolism, function and molecular biology of iron, copper, zinc and selenium. The format of the meet in follows that of previous FASEB conferences with slide presentations in the morning and evening, afternoons devoted to informal discussions/workshops, viewing of posters and recreation. Slide presentations will be delivered primarily by recognized experts with new results; presentations by younger scientists with rapidly developing projects will also be sought. Speakers will be directed to focus on their recent and most interesting findings. Two new sessions on knockout and transgenic animal models and molecular biology of human in-born errors will receive considerable attention. Regulation of gene expression by trace elements (2 sessions) reflects our increasing knowledge on the importance of trace element in gene regulation, as do two sessions on cellular functions of trace elements (2 sessions). Sessions on whole-body metal metabolism and new approaches to human trace element assessment will highlight recent human research. New molecular concepts will be featured in mechanism of absorption and metal cofactors sessions. In addition, four workshops will be organize to facilitate exchange of techniques and ideas on new molecular biology techniques, use of microorganisms as models, use of antagonists in models for mineral deficiencies, and mineral interactions. All participants will be invited to present posters; discussion leaders and workshops will be encouraged to bring attention to new observations reported in the posters. Today, the fields of molecular biology, biochemistry, nutrition, cell biology, medicine, immunology, pharmacology and toxicology all have active trace element components. The purpose of this conference is to bring this diverse group of individuals together to freely exchange results and ideas, to define important areas of needed research, and to develop novel schemes to solve both basic questions of biology and practical questions regarding interaction of nutrition and genes in human health and disease. The conference proceedings will not be published.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this research is to develop molecular biology-based assays for human selenium (Se) status. We were the first to report that mRNA levels of Se-dependent glutathione peroxidase-1 (GPX1) fall to 10% of Se-adequate levels, thus providing a molecular explanation for why GPX1 is an effective biochemical marker for assessing Se status in humans and animals, and suggesting the hypothesis that regulation of GPX1 expression is an important aspect of the role of GPX1. Using selenoprotein mRNA levels in liver as markers, we then carefully evaluated Se requirements in rats, showing that molecular biology markers were a useful tool for assessing requirements and that understanding the regulation of these molecular markers was important when assessing requirements throughout the life cycle, such as in pregnancy and lactation. More recently, we have conducted a series of studies investigating the use of blood (a less invasive tissue), and found that GPX1 mRNA expression in rat whole blood was comparable to levels in the major tissues and could be used effectively to assess Se status. Thus we have begun to evaluate human selenoprotein mRNA levels as a potential way to assess human Se status. The advantage is that these levels appear to be homoeostatic ally controlled by Se status. Secondly, the advent of rapid molecular biology assays suggests that molecular biology markers will soon become important in assessing human health, including human nutrition. We now have found that human blood, just as in rodent blood, has selenoprotein mRNA expression at levels comparable to major tissues, suggesting that these may be the effective markers of human Se status. In this proposed research, three specific aims will be pursued: 1. To assess selenoprotein mRNA expression using RPA on human whole blood from our current (unfunded) collaboration with Reading, England, to correlate these markers with plasma GPX3 activity and plasma Se levels; 2. To develop protocols and use quantitative real-time PCR (qRT-PCR) on human whole blood to assess selenoprotein mRNA expression for a number of selenoproteins in the Reading samples; 3. To assess whole blood selenoprotein mRNA levels, along with plasma GPX3 activity, RBC GPX1 activity, and plasma Se concentration, in 120 adult human subjects in Madison WI, and then to determine the changes in selenoprotein mRNA expression 1 mo, 2 mo and 4 mo after initiation of placebo or 110 ug Se per day supplementation in 20 male and 20 female subjects. Relevance: This research is directly focused on improving our understanding and ability to use specific molecular markers to set nutrient requirements, and thus is relevant to human health. More specifically, this research will directly address the research recommendations for selenium by the 2000 Institute of Medicine Dietary Reference Intake report, including the need for biomarkers for assessment of selenium status, by developing molecular biology-based assays that are linked to homeostatic regulation of selenium status and that have the potential to be used in individualized medicine. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This proposal requests support for the 8th International Symposium on Selenium in Biology and Medicine in Madison, Wisconsin, USA, on July 25 to July 30th, 2006, at the University of Wisconsin. The previous international symposiums in this series were held in Venice Italy (2000), Beijing China (1996) and Nashville USA (1992). An international organizing committee has been formed. 12 noted selenium researchers have agreed to chair sessions, and 30 speakers have agreed to give invited talks. The Wisconsin Union Conference Center has contracted to coordinate the registration, hotels, and abstract submission/program preparation. A website will cover recent advances in all areas of selenium biology and medicine. Oral sessions include: Selenoprotein Genomics; Se Incorporation into Selenoproteins; Characterization of Selenoproteins and their Function; Enzymes and Metabolites in Se Metabolism; Selenoprotein Knockout Models; Selenium-Virus Interactions; Human Se Metabolism and Requirements; Se and Cancer Chemoprevention; Se Metabolism and Speciation in Plants; Se in Domestic Animals; Bacterial Selenoenzymes and Unique Se Metabolism; Se in the Environment; Se and Reactive Oxygen and Nitrogen Species. A meeting size is projected to be 40 invited speakers and 230 registered participants, similar to previous meetings. Women and minorities will be well-represented as chairs and speakers, and special efforts will also be made to attract and support young investigators. The meeting starts with an opening session on Tuesday night, has 2 plenary sessions each morning, parallel oral sessions on 3 afternoons, poster sessions on 2 afternoons, an open afternoon to allow organized tours/activities, and a late afternoon final session on Saturday, followed by the banquet. The Memorial Union and its lakeside terrace is an idyllic setting that has come to be known and loved by all. Recent key advances in our understanding of selenium biology make this a timely meeting, and at the cutting edge in integration of molecular and genomic approaches to study of essential nutrients. The breath and depth of this meeting and its participants will help to better integrate this information, leading to additional advances and enhanced understanding of human health. [unreadable] [unreadable]