2008 - 2009 Alaska Heart Institute Fellowships
Danielle Kusmider (BS Biological Sciences): "Expression of MRF4 During Early Xenopus Development"
Faculty Mentor: Tim Hinterberger, WWAMI
Abstract: MRF4, Myf5, MyoD, and myogenin, the myogenic regulatory factors (MRFs), are a family of proteins expressed during skeletal muscle development, growth, and regeneration. Although MRF4 is known to function in the maintenance of the differentiated muscle cell type and is expressed during embryonic myogenesis, its role is incompletely understood. Current findings of MRF4 expression in Dr. Hinterberger's lab using Xenopus laevis, has led us to believe that the gene expression is not restricted to the myogenic cells. To further explore the expression of Xenopus MRF4, I will perform additional in situ hybridization on whole embryos brachial arches, otic vesicles, and head mesoderm. I will utilize different probe sequences coding whole-mount in situ hybridization specimens by comparing them to specimens sectioned prior to in situ hybridization. The findings will open doors to future studies of the transcriptional regulation of the MRF4 gene during differentiation of muscle and possibly other cell types. This will lead to better understanding of the regulatory function of MRFs and how genes are controlled.
Specific Aim #1: Determine tissue localization of specific cells of X. leavis that express MRF4 in the head and neck at early neural through metamorphic stages by sectioning specimens of whole mount in situ hybridization. Our recent findings of situ hybridization of whole mount X. laevis embryos contrdict what previous scientists have found. We plan to test and pin point the exact cells of X. laevis which express the MRF4 gene. Furthermore, we will determine whether our results are consistent when probes that correspond to different regions of the MRF4 gene sequence are used.
Specific Aim #2: Determine cell specificity in X. laevis expressing MRF4 in the head and neck at neural through metamorphic stages by section in situ hybridization. To assure that our probe is reaching and binding selectively to the target mRNA sequence of the specimen, we will perform section in situ hybridization. The intensity of staining will reveal whether any of the whole-mount staining is an artifact.
Project goals: The goal of this research project is to further understand the expression of MRF4 during early Xenopus development. This genetic study will present pioneering information about the transcriptional and signaling mechanisms controlling the expression of the MRF4 gene during specification and differentiation of muscle growth. By determining the specific cells expressing the MRF4 gene, we will gain a better understanding of the regulatory functions of the MRF's. This information will allow for further studies on how all genes are controlled and regulated.
Oksana Miller (BS Biological Sciences): Establishment of Nicotine and Cotinine Protocol Using Agilent LC-MS/MS; Determination of Vitamin D Levels in smokers and never-smokers
Faculty Mentor: Cindy Knall, WWAMI
Abstract: This study will provide an accessible and affordable means for future monitoring of tobacco use in Alaska and to determine the exacerbating effects of tobacco use on Vitamin D deficiency. Currently, there is no low-cost method available in Alaska for detecting nicotine levels in humans and tobacco and cigarette use in the State is common and particularly high among Alaska Natives. In fact, according to the National Health Interview Survey, the rate of cigarette smoking among Alaska Natives is almost twice higher than in the general US population. Alaska Natives have high rates of tobacco-related deaths and lung cancer remains to be one of the leading causes of all cancer-related deaths especially in rural regions of Alaska. Residents of Northern climates have found to have lower levels of Vitamin D than populations in southern or mid latitudes. Vitamin D deficiency has been linked to many forms of cancer, osteoporosis, diabetes, autoimmune diseases and heart disease. This second phase of this study will determine whether nicotine contributes to Vitamin D deficiency in Alaskans.
This study has two main aims: the first to establish a mass spectrometry protocol for detecting nicotine and its metabolites such as cotinine in human blood and urine samples for futur use in practices relevant to human health such as: monitoring smoking cessation efforts, detecting cigarette exposure in children and fetuses of smoking pregnant women, developing a tobacco control plan and also encouraging the smoking part of the population to reduce cigarette and tobacco use. The second aim is to determine whether nicotine and its metabolites contribute to Vitamin D deficiency in tobacco users.
Cody Rall (BS Biological Sciences): "Copper Homeostasis: Histone Modification at the CUP1 Locus"
Faculty Mentor: Jocelyn Krebs, Biological Sciences
Abstract: Cells maintain copper at specific levels because it is essential for many biochemical processes, yet toxic at high amounts. Copper homeostasis is maintained through various mechanisms that either uptake copper from the environment or sequester/export it from the cell. Genetic defects in copper homeostasis result in serious diseases in humans, including Menkes disease and Wilson’s disease. In the presence of high copper levels, the yeast Saccharomyces cerevisiae immediately expresses the CUP1 gene to produce the Cup1 metallothionein protein, which binds to excess copper within minutes of exposure. The CUP1 gene is then rapidly shut down within 30 minutes of copper exposure. Histone modifications, particularly acetylation, play a major role in controlling gene expression in all eukaryotes. I will investigate the pattern and function of histone modifications that coordinate this precise gene regulation, in order to understand the mechanisms controlling CUP1 activation and shutdown. I will also investigate histone acetylation patterns that may control expression of RUF5, an anti-sense RNA expressed from the complementary strand of CUP1. Finally, I will identify the enzymes responsible for these histone modification patterns. This proposal has two Specific Aims:
Specific Aim #1: To analyze the histone modifications that take place across the CUP1/RUF5 locus. This aim will take advantage of a powerful method, chromatin immunoprecipitation (ChIP), to precisely map the position and temporal regulation of specific histone acetylation events during copper exposure in living cells.
Specific Aim #2: To identify the enzymes responsible for histone acetylation patterns at CUP1/RUF5. Once the patterns of acetylation are established in Aim #1, I will use genetic methods to identify the roles of candidate enzymes in establishing these patterns, by testing the effects of deleting these enzymes on histone acetylation at CUP1/RUF5. I will test both enzymes that add acetyl groups to histones (lysine acetyltransferases; KATs) as well as enzymes that remove acetyl groups (histone deacetylases, HDACs).
Nicolette Skomp (BS Biological Sciences): Anaerobic and Aerobic Enzyme Activity in Harp and Hooded Seal Cardiac Tissue"
Faculty Mentor: Jennifer Burns, Biological Sciences
Marine mammal physiology has been well-studied in order to understand the adaptations that allow these animals to perform at intense levels in the absence of freely available oxygen. Bradycardia, vasoconstriction, and enhanced regulation of metabolic pathways are known to assist in prolonging oxygen availability during foraging and diving activities. However, the extent to which these mechanisms are utilized varies between skeletal and cardiac tissue. For example, while myoglobin (Mb), an oxygen storage protein is elevated in all marine mammal muscle tissues, it exists at higher levels in skeletal muscle than in cardiac tissue (Bishop, ongoing AHI funded research). This is likely in response to vasoconstriction during dives, in which skeletal muscle blood supply is drastically reduced in order to maintain constant perfusion of the heart and brain. Similarly, while it is known that seals are able to sustain aerobic metabolism during deep and long dives, the levels of aerobic and anaerobic enzymes that allow such activity are much better studied in skeletal tissue as compared to cardiac tissue. Here too, differences might exist. Skeletal tissue will likely employ anaerobic respiratory pathways as the oxygenation in peripheral tissue decreases, while cardiac tissue will continue to experience constant oxygen levels, and therefore will require higher levels of aerobic enzymes. Finally, studies have shown significant differences in metabolic capacities throughout marine mammal life span. In general, there are considerable increases in metabolic sophistication with age: for example, we have previously documented that Mb levels and acid buffering capabilities are markedly lower in seal pups in comparison to adults. Lower physiological capacity in pups likely reflects neonates’ low level of muscle development, which can be attributed to their young age and their low activity levels. By measuring enzyme activity in neonate cardiac tissue, we can get a better idea of the timeline of muscular metabolic development. The specific aims of this study are to measure enzymatic activity in cardiac tissue of pup and adult harp and hooded seals and compare results to that determined for skeletal muscle for the same animals. We will also compare results across species and age classes.
The first objective of this study is to test the hypothesis that seal hearts have elevated ability to generate ATP aerobically and reduced ability to generate ATP anaerobically relative to skeletal tissue due to constant perfusion of the heart during diving. To test this hypothesis we will measure three aerobic enzymes: citrate synthase (CS), isocitrate dehydrogenase (IDH) and β – hydroxyacyl-CoA dehydrogenase (HOAD), and two anaerobic enzymes: pyruvate kinase (PK) and lactate dehydrogenase (LDH).
The second objective of this study is to test the hypothesis that levels of aerobic and anaerobic enzymes in hearts will be lower in pups as compared to adults because of the decreased activity levels and limited diving abilities of young pups, as well as the lower Mb and buffering ability of their muscles. The data generated by this study will not only have important physiological and biochemical implications regarding the metabolic pathways of deep-diving marine mammals, but also may provide insight to the behaviors of immature seals as they develop from neonates to diving adolescents. These findings may also shed light on the development of human cardiac tissue and the behavior of human cardiac tissue in hypoxic conditions.
Alex Bonnecaze (BS Biological Sciences): "The Role of Leucine and Branched Chain Amino Acids in Regulating Protein Synthesis in Myotubes"
Faculty Mentor: Tim Hinterberger, WWAMI
Abstract: Leucine, isoleucine, and valine make up a distinct class of amino acids known as branched chain amino acids (BCAAs). BCAAs, especially leucine, have been shown to increase protein synthesis in skeletal muscle cells by activating the mammalian target of rapamycin (mTOR), a protein kinase. Leucine has also been show to directly increase the concentration of key initiation factors for protein synthesis, such as eIF4G. In addition to stimulating protein synthesis in muscle cells, leucine also slows protein degradation by binding to ubiquitin (Ub) proteasomes and preventing proteolysis. While leucine appears to be the main BCAA involved in increasing skeletal muscle protein synthesis, studies have determined that the addition of leucine without the other two BCAAs has a potential negative impact. The cellular presence of any of the three BCAAs activates branched chain ketoacid dehydrogenase (BCKD), an enzyme breaks down all three of the BCAAs. Due to the activity of BCKD it is generally believed that leucine should be administered in a proper ratio with the other BCAAs to avoid an imbalance. Muscle cells in a starved or exhausted state respond the most to the addition of BCAAs. While much has been learned about BCAAs' impact on skeletal muscle cells, many questions remain unanswered. Using C2C12 and L6 cells, two cultured cell lines that are widely studied models of normal skeletal muscle, we will determine what ratio of leucine to other BCAAs best stimulates protein synthesis. C2C12 and L6 myotubes will be tested in multiple trials involving the addition of BCAAs in different concentration ratios. Whether or not insulin synergistically works with BCAAs to increase protein synthesis will also be tested. We will also determine if leucine has any effect on the level of the myogenic factor MRF4 protein, because increased MRF4 has previously been found to be associated with muscle growth. If MRF4 protein is increased, the possibility that these treatments increase levels of MRF4 gene transcripts will be examined.
Aim #1: Compare how C2C12 (mouse) and L6 (rat) muscle cell lines respond to the administration of leucine/BCAAs Administration of leucine alone increases BCKD activity, potentially causing an amino acid imbalance and negatively impacting the cell. We plan to test how total cellular protein levels vary depending on the ratio of BCAAs administered to C2C12 and L6 cells. Insulin’s effects in conjunction with leucine will also be examined. Protein levels will be measured via spectrophotometric protein assay.
Aim #2: Determine if MRF-4 protein level increases with leucine/BCAA concentration. After the best culture conditions and cell line with the best protein response to leucine is determined, we will see if MRR-4 protein level increases in response to the addition of leucine. Western blot with a commercial anti-MRF4 antibody will be used to determine levels of MRF-4.
2007 - 2008 Alaska Heart Institute Fellowships
Trevor Ray Thomas (BS Biological Sciences): "Role of Williams Syndrome Transcription Factor in Xenopus laevis"
Faculty mentor: Jocelyn Krebs, Biological Sciences
Abstract: Willimas Syndrome (WS) is a genetic disorder that results in cardiovascular defects and visual/spatial processing deficits. One of several genes absent in WS patients is Williams Syndrome Trascription Factor (WSTF). WSTF interacts with Imitation Switch (ISWI) to form the WSTF-ISWI Chromatin Remodeling Complex (WICH), an ATP-dependent chromatin remodeling complex that is conserved in vertebrate species. When WSTF expression is inhibited in vivo in Xenpus laevis embryos, the WICH complex is destabilized, and the resulting embryos exhibit defects in development of the brain and eye. We have begun to investigate the effects of WICH complex depletion on two key genes involved in neural development: bone morphogenic protein 4 (Bmp4) and Sonic Hedgehog (Shh). Bmp4 antagonizes neural development, and its levels are reduced during early neurulation in normal Xenopus embryos, though Bmp4 is later required during development of the eye. In contrast, Shh is essential for development of neural tissues. Shh levels in whole embryos decrease in the absence of either WSTF or ISWI, and Bmp4 levels increase in the absence of ISWI. However, both Bmp4 and Shh are expressed in precise spatial patterns in developing embryos, and it is not known whether depletion of WSTF or ISWI changes the patterns of expression of these genes. I will use in situ hybridization to map the spatial and temporal expression of Bmp4 and Shh in embryos lacking WSTF. I will also test whether specific conserved regions of the WSTF protein are required for normal expression of Bmp4 and Shh.
2006 - 2007 Alaska Heart Institute Fellowships
Stephen Wolfe (BS Biological Sciences): "Does a normobaric hypoxia sleep regimen affect work performance at high altitude?"
Faculty Mentor: C. Loren Buck, Biological Sciences
Abstract: The military frequently sends service personnel into extreme environments, including high altitude, on short notice. At high altitude, unacclimatized personnel experience a reduction in work capacity, and risk altitude-related illnesses. A few military units have implemented nomobaric hypoxia training (NHT), intermittent exposure to low ambient oxygen levels at normal barometric pressure, to prepare for high altitude deployments, NHT (via exercise or sleep regimen) is effect in enhancing sea level performance, but its usefulness for acclimatization has not been established. The Alaska Air National Guard's 212th Rescue Squadron has recently purchased normobaric hypoxia equipment to enhance its ability to meet its mission requirements, but lacks a training protocol for its use. In collaboration with U.S. Army Research Institute for Environmental Medicine (ARIEM), we will investigate effects of a 10-day sleep hypoxia regimen on work performance under normobaric (sea level) and hypobaric (high altitude) hypoxia conditions.
We will assess work performance via a 500 Kilojoule bicycle ergometry time trial; the time to complete 500 Kj of work before and after the 10-day treatment or control regimen. In addition to performance assessment, we will collect data on blood gas and chemistry from all subjects to compare hematological responses between treatment and control groups. Blood data will establish reference values for continuing studies of physiological and medical implications of high altitude deployments and treatments and serve to inrease our mechanistic understanding of high-altitude acclimatizaation. The knowledge gained from this study will not only enhance our unit's capabilities and safety, but also may increase our knowledge and efficacy in patient care at high altitudes.
Nancy Bishop (BS Biological Sciences): "Myoglobin Concentrations and Acid Buffering Capacity in Seal Cardiac Tissue"
Faculty Mentor: Jennifer Burns, Biological Sciences
Abstract: Aquatic mammals, particularly those who participate in deep diving behaviors, require different physiological and biochemical adaptations to survive in low oxygen environment. These animals have been shown to have increased lung capacity, higher blood volume, higher myglobin and hemoglobin concentrations, and larger muscle mass, compared to terrestrial animals (Kooyman & Ponganis, 1998). These variations allow the animals to sustain aerobic respiration for longer periods of time while breath-hold diving. Hooded and harp seals can both make long, deep dives that require adaptations promoting oxygen conservation, as well as those which allow for anaerobic respiration byproducts, such as lactic acid, to be buffered so that tissue pH levels do not change substantially. The physiological and biochemical adaptations that promote breath-hold diving have been studied in skeletal muscle, yet little research has been done to determine adaptations of the cardiac muscle. Cardiac muscle has a greater requirement for sustained oxygen availability than skeletal muscle because the former cannot resort to anaerobic respiration (Kooyman & Ponganis, 1998). In light of this, we would expect that the myoglobin content and acid buffering capabilities would differ between these two tissues. Furthermore, neonatal seals are largely terrestrial for their first several weeks, and the absence of diving in these young animals may negate the need for increased oxygen preserving mechanisms. However, as the seals near maturity and begin to participate in deep diving activities, their physiology must develop in order to accommodate for the lack of oxygen underwater.
In order to determine the pattern of physiological development in young seals, our project will specifically examine the concentrations of myoglobin and acid buffering capabilities in the cardiac muscle of 27 seals over the 2007 summer and fall semesters. Comparisons will be made within species between age groups, as well as between similar aged individuals in the two species. Differences in myoglobin content and acid buffering ability of cardiac and skeletal muscle tissue will also be examined. This research will reveal how adaptations for withstanding periods of low oxygen availability develop in the heart tissue of these two deep-diving seal species. This information in turn may give us more insight into the abilities of the cardiac muscle to withstand short periods of hypoxia, such as frequently occur in humans when blood vessels providing oxygen to cardiac tissue are blocked.
Mindy Graham (BS Chemistry): " Panton-Valentine leukocidin expression in community associated methicillin resistant Staphyloccocus aureus from rural Alaska"
Faculty Mentor: John Kennish, Chemistry
Abstract: Incidence of infection by Methicillin resistant Staphyloccocus aureus (MRSA) has historically been attributed to nosocomial sources, but there is currently an increasing trend towards community acquired infection not associated with hospital settings. Community-associated MRSA (CA-MRSA) is most often associated with skin and soft tissue infections rather than invasive disease. The majority of isolates causing skin infections in healthy peoplecarry a gene for a toxin called Panton-Valentine leukocidin (PVL). Presence of the PVL gene in the majority of community associated human skin infections has led to general acceptance for PVL as an important virulence factor. One recent study, however, has suggested that presence of the PVL gene has no effect on MRSA virulence and so this has become a point of contention in the understanding of the pathogenesis of these infections. The objective of this experiment is to verify if PVL is indeed a virulence factor in community associated-MRSA (CA-MRSA) by studying isolates collected from a skin infection outbreak in rural Alaska.
This experiment will detect and quantify the expression of PVL by studying messenger ribonucleic acid (mRNA). PVL mRNA levels will be measured using reverse transcriptase real-time polymerase chain reaction (RT-PCR). I am hoping to determine whether or not CA-MRSA strains that carry the PVL gene are actually producing the PVL toxin. If findings show that the PVL gene is universally expressed, then it can be suggested that PVL plays a role in the pathogenicity of CA-MRSA skin infections. If PVL expression levels differ between isolates of the same outbreak, this might provide supporting evidence for an alternate mechanism of infection.
Blaine Shillington (BS Engineering): "Construction and Validation of an In Vitro Exposure System"
Faculty Mentor: Cindy Knall, WWAMI
Abstract: This project proposes to research the effect of atmospheric pollutants in cigarette smoke on live respiratory cells. I will be assisting in this study by helping design and modify a pre-existing plan, and then by assembling said design. This design system will then be used as an exposure system to study the controlled exposure of smoke and nicotine on live cells. I will be responsible with the assembly and testing of the exposure system, before laboratory testing can begin.