Progress 09/15/06 to 09/14/09
Outputs
OUTPUTS: Outreach and dissemination is provided through the Agricultural and Forestry Experiment Station publications website: http://www.uaf.edu/salrm/afes/. FIELD CULTIVATION: Successful seed germination protocols for rapid propagation by seed were developed. Successful protocol for stem cutting propagation of V. uliginosum was developed and the variability in rooting by site and from plant to plant was explored. This information was shared directly with individual berry growers, members of the Alaska Berry Grower Association and the general public at a regional conference, Grown in Alaska, Palmer, Alaska 2009. A scientific poster was presented at the American Society for Horticultural Sciences national meeting, St Louis, MO, the International Plant Propagators' Society meeting, Denver CO and a public interest leaflet was revised to reflect recent research. Three universities are collaborating with us on a Specialty Crop proposal. CONTROLLED ENVIRONMENTS: A graduate student, Heidi Rader completed her M.S. A publication has been submitted to Acta Horticulturae. CHEMICAL ANALYZES: The graduate student on this project (Colin McGill) has essentially completed the research obligation of this PhD project. It is expected that he will complete this thesis and defense and graduate by Spring 2010. Associated with this goal, the structures and activities of the isolated compounds will be submitted for publication in the Journal of Natural Products. The authorship will include Colin McGill (grad student), Sigourney Walker (undergraduate research student) and possibly Jennifer Chambers (undergraduate research student). INTERACTION OF ANTI-OXIDANTS AND CONTAMINANTS: A high school teacher received training on the ELISA technique which she will use with her students on high school research projects. PARTICIPANTS: PARTICIPANTS: Dr. Carol E. Lewis, Director AFES, Project Oversight. FIELD CULTIVATION:Dr. Patricia Holloway, Professor of Horticulture, Grant Matheke and Jan Hanscom, Research Technicians. I visited members of the Alaska Berry Growers Association, Kenai and shared information on berry production and propagation with commercial berry producer, Brian Olsen, Kenai. I continued connections with growers in Prince of Wales Island who have successfully propagated V. ovalifolium by air layering. Two growers in the Interior, Charles Knight and Papa Meneur, propagated thousands of seedlings of V. vitis-idaea and V. uliginosum and have transplanted them into cultivated fields. Two graduate students, Tina Buxbaum, and Rehanon Pampell, are working on pollination biology and bumble bee biology to enhance fruit set in wild and cultivated stands using native pollinators. We have developed a collaborative team with USDA ARS entomologist, Dr. Alberto Pantoja, UAF entomologist, Dr. Derek Sikes, and Alaska beekeeper, Stephen Peterson, to expand knowledge of native pollinators. CONTROLLED ENVIRONMENTS:Meriam Karlsson, Professor of Horticulture, project manager; Jeff Werner, Research Professional; Jeffrey Johnson, Research Associate; Yosuke Okada, graduate student; James Ward, B.S. student; Andrew Winkelman, B.S. student; Patrick Sanders, B.S. student; Chena Hot Springs Resort, Collaborator, Pike's Waterfront Lodge, Collaborator, Rough Brothers Greenhouse Manufacturing Company (Cincinnati, Ohio), Collaborator, Link4 Corporation (Placentia, California), Collaborator, Svensson Screens (Charlotte, North Carolina), Collaborator CHEMICAL ANALYSIS: Dr. Thomas Clausen, Department Chair and Professor of Chemistry and Biochemistry. The success of this last year's research is largely due to the collaboration between Dr. Kuhn's and Dr. Clausen's labs. Colin McGill (PhD student) performed most of the extractions, fractionations, and structure determinations. His lab work is nearly completed and he is in the process of writing up his thesis. He is expected to graduate this academic year. Sigourney Walker (Undergraduate Chemistry Student) participated in many of the isolations including IV, fructose, and cyaniding. Talal Ahmed, Gavin Baker, Patrick Bollig, Jennifer Chambers, Carrie Duplisea, Christopher Ebner, Kathleen Gerdes, Andy Lester and Brad Weis (Organic Laboratory {Chem 324W} students) performed the first two steps in the synthesis described in Figure 2. CHEMICAL INTERACTIONS: Dr. Duffy, a professor at UAF, led the project while Dr. Dunlap, a research assistant professor, managed and maintained the lab and cell cultures. A high school teacher received training on the ELISA technique which she will use with her students on high school research projects. A graduate student also participated in the design of the modifications for year 2 of this project. COLLABORATORS: Controlled Environment: Chena Hot Springs Resort, Pike's Waterfront Lodge. Chemical Analyses: Dr. W. Kalt (Agriculture Canada) and the Wild Blueberry Association of North America (Director S. Davis) Professor Marina Castillo, Pharmacology and Toxicology TARGET AUDIENCES: Owners, managers, employees of local greenhouse, horticulture operations and businesses; individuals considering potential commercial ventures; students; researchers, Alaska natives. FIELD CULTIVATION: The users of the berry information range from home gardeners who are interested in growing bog blueberries in edible landscapes to small cottage industries based upon processed jam and jelly products. It includes the Alaska Berry Growers Association, Alaska Blues. CONTROLLED ENVIRONMENTS: Owners, managers and employees of state-wide commercial greenhouse and other horticulture operations and businesses; berry and specialty crops growers, individuals considering or participating in horticulture production ventures; students in secondary and post-secondary educational programs and college level undergraduate and graduate students; initial and continuing training opportunities for the local workforce of horticulture operations, plant physiologists, climate change and climatology researchers. CHEMICAL INTERACTIONS: The incidence of brain cancer in Alaska is higher than the national average. This research on cancer and neuroprotection is very important to Alaska natives and all people living in the circumpolar north. The understanding of protective mechanisms of berries, a local food, is important for the berry and neutraceutucal industry. Another audience is the newly developing biotech industry in Alaska. PROJECT MODIFICATIONS: FIELD CULTIVATION:The focus of this research has been narrowed to include two species of Vaccinium, V. vitis-idaea and V. uliginosum, in part because of requests from constituents and also because of climactic challenges working in Interior, Alaska. Some of the coastal species of Vaccinium cannot survive in Interior, Alaska, and I have not found a suitable location for field trials in coastal Alaska. The two remaining species have the greatest economic potential in Alaska. Whereas, the species have been narrowed, the scope of the project has expanded to include important components to management of wild and cultivated stands including methods of field establishement and pollination biology for improved fruit production. Both of these areas were pursued at the request of the Alaska Berry Growers Association.
Impacts
FIELD CULTIVATION: Bog blueberry fruit, Vaccinium uliginosum, were collected. Experiments were conducted to compare germination between air dried seed and seed from frozen berries. We found that optimum germination occurs with seeds extracted from frozen berries and sown immediately and not permitted to dry out. Stem cuttings of V. uliginosum:Softwood stem cuttings of wild-harvested bog blueberry from new growth rooted more than 50% from 20 June through August, and rooting did not differ among collection dates. Cuttings collected from 30 different wild locations showed a significant location effect in rooting percentages. All rooted cuttings overwintered in the greenhouse survived and began growth in April 2008. CONTROLLED ENVIRONMENTS: A retractable double-layer flat-roof high tunnel (Rough Brothers, Cincinnati, Ohio) has been erected. The total covered field area is 11 by 18 m (36 by 60 feet) with a post height of 3.7 m (12 feet). The tunnel is equipped with one layer of translucent material for crop production and a blackout covering for photoperiodic control. CHEMICAL ANALYZES: Guided by the NADPH oxidase and sMas assays run in Professor Kuhns laboratory, we isolated five active compounds from Alaska bog blueberry in addition to one inactive compound (fructose) and one untested compound (cyanidin). The active compounds were surprisingly diverse in structure. Four of the compounds are best classified as secondary metabolites (1-IV) while V is clearly a primary metabolite. Of the secondary metabolites, two are from the terpenoid pathway (I & II), one from the shikimate pathway (III), and one (IV) either from the acetogenin pathway or perhaps an offshoot of a primary metabolic pathway involving malic acid. INTERACTION OF ANTI-OXIDANTS AND CONTAMINANTS: In our USDA funded project we developed bioassays that identified both antioxidant and anti-inflammatory properties of blueberry extracts and isolated fractions using the TAP assay and sphingomyelinase assay.
Publications
- Duffy, L.K., Dunlap, K.L., and Nicholas-Figueroa . 2009 Chemo-protection for brain cancer by Alaska blueberries. 2009 Arctic Science Conference abstract.
- Holloway, P. S., K.M. Kokx, J. Auer and S. Pearce. 2009. Propagation of Alaska bog blueberry, Vaccinium uliginosum. HortScience. 44(4):1102. Abstr.
- Holloway, P. 2009. How to germinate seeds of Alaska wild blueberries and lingonberries (lowbush cranberries). Georgeson Botanical Notes No 34. (revised) 2p.
- Karlsson, M. and J. Werner. 2010. High tunnel covering materials for northern field production. Acta Horticulturae
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Neuroprotection: Utilizing a high-throughput fluorescence assay, we demonstrated that either aqueous or organic extracts prepared from Alaska Wild Bog Blueberries (Vaccinium uligunosum) contain a potent inhibitor of nSMase. We did not found a significant difference among Alaska Wild Bog Blueberries collected from three distinct geographical locations in the interior of Alaska (Lake Minchumina, Fairbanks, Fox). Most importantly, the inhibitory activity had no antioxidant properties at the concentrations used. Chemical Analysis: Further fractionation of blueberry extracts guided by our high throughput fluorescence nSMase assay resulted in a significant enrichement of the inhibitory compound. Thin layer chromatography revealed a small number of compounds in most fractions including the inhibitiory fraction and sufficient material was obtained to perform NMR analysis. This project has supported two undergraduate research students over the last year. Amy Rask was a senior chemistry major and met her undergraduate research requirement last spring by helping to isolate and characterize the active fractions. Last summer, Meghan Sanford obtained undergraduate research credit for pursuing similar work. Both of these students have been awarded the department's Outstanding Chemistry Student award. This project has also supported a PhD graduate student in Biochemistry, Colin McGill, throughout his graduate studies. He is on track to graduate in May 2009 and will submit his research results, including the results that are briefly described in this report, disseminated in a nationally peer-reviewed journal. PARTICIPANTS: Dr. Carol E. Lewis, Director AFES, Project Oversight. Field Cultivation: Dr. Patricia Holloway, Professor of Horticulture, Grant Matheke and Jan Hanscom, Research Technicians. Controlled Environment: Dr. Meriam Karlsson, Professor of Horticulture and Jeff Werner, Research Professional, Terry Marsh, Research Associate, Heidi Rader, M.S. student; Yosuke Okada, James Ward, Andrew Winkelman, Patrick Terra, Patrick Sanders, Justin Hogrefe, Melissa Gagnon, kate Fournier, David Aguiar, Shawn Biessel, Kalan Paul, Mia Peterburs, Ryan Sloger B.S. students. Chemical Analysis: Dr. Lawrence Duffy, Chemistry Department Head, Project Oversight. Dr. Tom Kuhn, Assistant Professor of Chemistry and Biochemistry, Dr. Kriya Dunlap, Research Professional, Dr. Thomas Clausen, Department Chair and Professor of Chemistry and Biochemistry. The success of this last year's research is largely due to the collaboration between Dr. Kuhn's and Dr. Clausen's labs. Dr. Kuhn students: Brian Barth, S.J. Gustafson, and Sally Brown, PhD graduate research students. Collaborators: Controlled Environment: Chena Hot Springs Resort, Pike's Waterfront Lodge. Chemical Analyses: Dr. W. Kalt (Agriculture Canada) and the Wild Blueberry Association of North America (Director S. Davis) Professor Marina Castillo, Pharmacology and Toxicology TARGET AUDIENCES: Owners, managers, employees of local greenhouse, horticulture operations and businesses; individuals considering potential commercial ventures; students; researchers, Alaska natives. EFFORTS:The project has also resulted in several student presentations at local, regional, and national events along with a joint publication with two PIs and three graduate students: Invitation to three presentations (Tufts University, University of Montana, Winter Conference on Brain Research; Press Conference at the Annual Meeting of the Society for Neuroscience; Three manuscripts in preparation for submission: i) describing the NOX inhibitiory activity of AK wild blueberries, ii) elaborating on the protection of actin oxidation by nSMase inhibition via AK wild blueberry compounds, and iii) a review on NOX inhibitor currently indentified; Two NIH R01 grants submitted:Exploring the Antagonistic Effects of Dietary Antioxidant Supplements on the Brain Health Benefits of Berry Polyphenols Donald K. Ingram (PI), and James A. Joseph and Thomas B. Kuhn (Co-PI) AND The effects of blueberry polyphenols in reducing sphingomylenase-induce oxidative and inflammatory stressors in aging James A. Joseph (PI) and Thomas B. Kuhn (Co-PI) PROJECT MODIFICATIONS: Subproject: Elucidating the neuroprotective role of compounds isolated from blueberries 1. To continue to determine the antioxidant efficacy of blueberry extract or polyphenols using ORAC and TAP assays with a focus on regional variation and level of contaminants 2. To determine the interaction of blueberry extracts and polyphenols in the presence of mercury and PCB by measuring CRP and IL-6 in a cell culture system.
Impacts
Wild Alaska Blueberries, in particular, contain exceptionally high levels of polyphenols. Our long-term goal is to elucidate 1) the molecular mechanisms confering neuroprotection against inflammatory processes and oxidative stress by natural products in Alaska Wild Blueberries and 2) to characterize these natural compounds as a lead drug development approach to enhance therapeutic or preventative strategies to alleviate and/or adverse effects of neuroinflammation. The inhibitory activity was reported in Current Topics of Nutraceutical Research. This research was further highlighted at a special Press Conference at the 2007 Annual Meeting of the Society for Neuroscience. Thus far we have two compounds identified by NMR that need verification by LC-MS/MS. These experiments would demonstrate for the first time a natural compound from Alaska Wild Bog Blueberries, which 1) is beneficial in combating inflammation in the nervous system, 2) specifically interacts and inhibits a molecular target in neuronal cells, and 3) could serve as a lead drug/nutraceutical development study.
Publications
- Gustafson SJ, Barth BM, McGill CM, Clausen TP, Kuhn TB. 2007. Wild Alaskan blueberry extracts inhibit a magnesium-dependent neutral sphingomyelinase activity in neurons exposed to TNFa. Current Topics in Nutraceutical Research 5(4):183-188)
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: CONTROLLED ENVIRONMENT: Research data is being collected on plastic covering materials for field crop production. Of the four cultivars of strawberries, Fern, Tristar, Tribute, and Quinault, Tristar and Tribute appear to outperform in all locations. Tulameen raspberries continue to result in high fruit yields. FIELD CULTIVATION: Work continues with seed germination and field cultivation of wild blueberry species. Work with coastal blueberries has been limited because of lack of plant materials. Presently, we have 10 jars in culture on V. ovalifolium. The plant does not produce microshoots as readily as V. uliginosum and V. vitis-idaea. We will begin an experiment with zeatin and 2-iP in November 2007. CHEMICAL ANALYSIS: The proinflammatory cytokine tumor necrosis factor (TNF) is key to initiating and orchestrating inflammation, which substantially contributes to the progression of many chronic and acute CNS pathologies. TNF can stimulate a magnesium dependent neutral
sphingomyelinase resulting in the accumulation of ceramide, a lipid messenger implicated in oxidative stress and apoptosis. Dietary polyphenols were shown to alleviate CNS inflammation largely attributed to their antioxidant properties. We found that preincubation of human SH-SY5Y neuroblastoma cells with organic or aqueous extracts prepared from Alaska wild bog blueberries completely negated Mg2+-nSMase activation upon TNF exposure. This specific and potent inhibition of Mg2+-nSMase activity was non-antioxidant in nature.
PARTICIPANTS: Dr. Carol E. Lewis, Director AFES, Project Oversight. Controlled Environment: Dr. Meriam Karlsson, Professor of Horticulture and Jeff Werner, Research Professional and Heidi Rader, M.S.student. Field Cultivation: Dr. Patricia Holloway, Professor of Horticulture, Grant Matheke and Jan Hanscom, Research Technicians. Chemical Analysis: Dr. Lawrence Duffy, Chemistry Department Head, Project Oversight. Dr. Tom Kuhn, Assistant Professor of Chemistry and Biochemistry, Dr. Thomas Clausen, Department Chair and Professor of Chemistry and Biochemistry. The success of this last year's research is largely due to the collaboration between Dr. Kuhn's and Dr. Clausen's labs. This interaction has allowed Colin McGill,PhD student, to fractionate blueberry extracts into purified components of biological (nutraceutical) interest. Dr. Kuhn students: Brian Barth, PhD graduate student and Sally Brown, undergraduate research student, who is entering graduate studies, has provided the bioassay.
TARGET AUDIENCES: The target audiences for field cultivation and controlled environments are entrepreneurs interested in large scale berry production. The target audiences for the chemical analysis of blueberries are those interested in the pharmaceutical and nutraceutical properties of high latitude grown berry crops.
Impacts
The future impact will be economic opportunity that will enhance the quality of life for Alaska's people and communities. Our goal of sustainable and responsible development of Alaska's abundant supply of wild- harvested berries is being realized. Alaskan grown crops are high in antioxidants exceeding levels of similar berries found at lower latitudes. Research is showing that Alaska grown blueberries have a inflamation inhibiting substance which researchers are working to identify. This project continues the work begun in ALK-06-01 ACCESSION NO: 0205028.
Publications
- No publications reported this period
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