Dr. Timothy Royappa

Dr. Venkat Sharma

Dr. Ted Fox

Dr. Glenroy "Dean" Martin

Background and Significance:  

Hypothesis and Objectives: Discorea bulbifera synthesizes important compounds that facilitates its rapid growth rate and may modulate its biological activity. In analyzing extracts from D. bulbifera, we have found some fractions that inhibit seed germination and bacterial growth in our bioassay systems. Here, we propose to extend these findings through the following objectives: 1). Isolate and identify brassinosteroids and related compounds from several distinct native populations of D. bulbifera in the Florida panhandle. 2). Evaluate the ability of the isolated compounds to regulate plant growth in vitro. 3). Screen the isolated compounds for biological activity in non-plant systems.

Methodology: Objective 1: Tissues samples will be collected at field sites in Franklin and Jackson Counties. Once transported to the lab, samples will be lyophilized and extracted. Extracts will be analyzed via thin layer chromatography (TLC) and purified on normal and reverse phase HPLC. The structures of pure isolates will be elucidated by 1- and 2-dimensional NMR, MS and other spectroscopic and chemical means. Objective 2: D. bulbifera will be introduced into tissue culture allowing evaluation under strictly defined growth conditions. Compounds isolated in Objective 1 will be introduced to the cultures at a range of concentrations and evaluated for their ability to stimulate biomass production and shoot and root proliferation. Objective 3–The isolated compounds will be screened in a biological assay for in vitro anti-fungal, anti-bacterial, and insecticidal activity using Colletotrichum gloesporiodes (plant pathogenic fungus), Penicillium chrysogenum, Escherichia coli, and Drosophila melanogaster.

Dr. Michael Huggins

Background and Significance:  myo-Inositol, one of nine positional isomers of inositol, is relatively abundant in organisms ranging from bacteria to man. Functionally, the role played by phospho-myo-inositides (phospholipids containing a myo-inositol headgroup) in trans-membrane signaling pathways has been well studied. Other inositols such as scyllo-, chiro-, and neo-inositol have been detected in various organisms including Tetrahymena, a unicellular eukaryotic protist. In addition to myo-inositol, these cells appear to contain significant quantities of scyllo-inositol, some of which is found in the phospholipid fraction. In general, the overall biological significance and functional role of free scyllo-inositol and scyllo-inositol-containing phospholipid remain to be systematically investigated.

Hypothesis and Objectives:  The proposed research is predicated on the hypothesis that fluctuations in the intracellular concentrations of scyllo-inositol and phospho-scyllo-inositides result in Tetrahymena undergoing temperature stress or morphological transformation. Preliminary studies have indicated that free scyllo-inositol concentration increases in Tetrahymena cells subjected to chilling stress. These data suggest that a subsequent increase in phosphatidyl-scyllo-inositol also might occur. Should such changes be shown to occur, they would be strongly indicative of a functional role. The objectives of this project are to: 1) prepare total free inositol and total phospholipid extracts from Tetrahymena under control conditions and conditions of temperature stress and during various stages of morphological transformation (certain species of Tetrahymena can be easily induced to convert between small and large oral apparatus phenotypes under controlled laboratory conditions) and 2) identify and quantify the free inositol isomers and phospholipids present in the respective fractions by NMR techniques.

Methodology:  Samples will be prepared by extracting cells in acidified chloroform/methanol. A total phospholipid fraction will be prepared from the organic phase of the extraction by preparative thin-layer chromatography on silica gel. A total free inositol fraction will be prepared from the aqueous phase by strong anion exchange chromatography. NMR will be performed using a 300 MHZ Varian MercuryPlus NMR spectrometer. ³¹P NMR of the phospholipid fraction will follow the methods described by Meneses and Glonek. ¹H/¹³C NMR of the inositol fraction will follow standard methods based on chemical shift estimations and molecular symmetry. Two-dimensional NMR techniques (¹H NMR) will be used to verify the chemical shift assignments necessary to quantify the amounts of the various inositol isomers in the samples. There will be some method development required in order to perform the analysis of the inositol and phosphoinositol isomers via NMR.

Dr. Timothy Royappa

Background and Significance: Cytokines are small peptides intricately involved in immune regulation. We wish to exploit the potentially enhanced avidity offered by novel multivalent polymer-cytokine conjugates by synth­esizing such bioconjugate materials. We have been active in identifying several B-cell lymphokines that could interrupt viral induced cytokine modulations. It is our hope to couple cytokine ligands to a hyperbranched polymer scaffold to block viral induced cytokines as a pharmaco­logical strategy to regulate immune function. Prior work has shown that, e.g., PEG-interleukin con­jugates have biological properties superior to those of unconjugated interleukins. Our premise is that multivalent bio­conjugates will possess yet more enhanced properties such as increased bioavailability of circulating levels of cytokines.

Hypothesis and Objectives:  Multivalent attachment of cytokines to a hyperbranched polymer leads to enhanced immunomodulation. Synthesis and evaluation of novel hyperbranched polyether-cytokine complexes.

Methodology:  Polyglycidol is a cheap, hyperbranched, water-soluble polyether with num­erous terminal hydroxyl groups, and may be considered a highly branched analog of poly­ethylene glycol (PEG). In vitro studies from our laboratories on human B-cell lines have shown that polyglycidol shows no cytotoxicity, similar to PEG, at various concentrations. The -OH groups of polyglycidol can be activated with sulfonyl chlorides, followed by ligand coupling, as shown below.

Activation:      Polymer-(OH)_n  +  n R-SO₂Cl  ®  Polymer-(O-SO₂-R)_n  +  n HCl

{R = -CH₂CF₃ (2,2,2-trifluoroethanesulfonyl chloride) or -C₆H₄CH₃ (p-toluenesulfonyl chloride)}

Coupling:        Polymer-(O-SO₂-R)_n  +  n H₂N-Ligand  ®  Polymer-(NH-Ligand)_n  +  n HOSO₂R

Polymer-(O-SO₂-R)_n  +  n HS-Ligand  ®  Polymer-(S-Ligand)_n  +  n HOSO₂R  

We have carried out this activation and coupling with a model protein, bovine trypsin, and have found that the result­ing trypsin-polyglycidol conjugate could be easily purified with size-selective centrifugal filter devices. How­ever, we had difficulty assaying the biological activity of these conjugates, and the molecular weight of the polymer was also rather low (Mn » 1000 g/mol). Our present efforts are centered on increasing the molecular weight of this polymer by controlled thermal crosslinking (manuscript in preparation) and on coupling with a different model protein, horse­radish peroxidase, that is more easily assayed. We will subsequently extend this work to other ligands such as cytokines.

Dr. Wade Jeffrey

Dr. Pam Vaughan

Background and significance: Climate change manifesting in ocean acidification, increased temperature and higher UV levels through stratospheric ozone depletion has fueled recent research interest into the effects of these factors on the organisms responsible for critical biogeochemical cycles. Recent work examined the photo-protection response of phytoplankton cultures to increased UV exposure through the production of mycosporine-like amino acids (MAAs) and their ability to produce these under nitrogen limited conditions. It is important to understand this protection response and how natural variations in UV exposure, temperature, salinity and nutrient availability influence the efficiency of this process.

Hypothesis and Objectives: This study will investigate the seasonal and spatial variations in the UV absorbing capacity of natural phytoplankton communities through quantification of various pigments associated with photo-protection (carotenoids and MAAs). Objective 1) Optimization of pigment analysis methodology, 2) Data analysis relating seasonal patterns in pigment production and other variables such as UV exposure, temperature, salinity and nutrient availability, and 3) Determine if pigment production can be induced through exposure to light from various regions of the UV spectrum.

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