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Conopeptidomics of Conus regius
Title
Conopeptidomics of Conus regius.
Creator
Franco, Aldo, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
The main objective of this dissertation is the isolation and characterization of novel neuroactive peptides from Conus regius. The conopeptides targeted in this work have a MW of 3500 Da or less, in the hopes that they can become viable drug candidates. A total of 30 sequences were isolated and characterized from the venom of Conus regius, giving us a partial library of the conopeptides found in this species. Techniques such as size exclusion chromatography, reversed phase chromatography,...
Show moreThe main objective of this dissertation is the isolation and characterization of novel neuroactive peptides from Conus regius. The conopeptides targeted in this work have a MW of 3500 Da or less, in the hopes that they can become viable drug candidates. A total of 30 sequences were isolated and characterized from the venom of Conus regius, giving us a partial library of the conopeptides found in this species. Techniques such as size exclusion chromatography, reversed phase chromatography, mass spectrometry, nano-nuclear magnetic resonance, chemical modifications of peptides, peptide sequencing through Edman degradation and in some instances bioassays were used together in an effort to perform "conopeptidomics" of Conus regius. The first chapter deals with Conus regius M-superfamily conopeptides. The second chapter is about the A-superfamily conopeptides found in Conus regius. The third chapter deals with Conus regius P-superfamily conopeptides. Finally the fourth chapter encompasses the T-superfamily conopeptides and all other small and linear peptides found in Conus regius that do not have a classification. This work is the first example reported, for any cone snail species, where most of the components of the venom have been sequenced directly for a single cone snail species. This work shows that a more realistic library of conopeptides can be obtained by direct analysis of the venom as opposed to cDNA libraries, which while useful; it does not reflect the post-translational modifications commonly found in conopeptides.
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Date Issued
2006
PURL
http://purl.flvc.org/fcla/dt/12206
Subject Headings
Chemistry, Biochemistry
Format
Document (PDF)
Diterpene biosynthesis in early ontogenetic stages of Pseudopterogorgia elisabethae and Pseudopterogorgia bipinnata
Title
Diterpene biosynthesis in early ontogenetic stages of Pseudopterogorgia elisabethae and Pseudopterogorgia bipinnata.
Creator
Mukherjee, Maia Stapleton, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
There is growing evidence that secondary metabolites isolated from some marine invertebrates may actually be produced by a symbiont. The capacity of early ontogenetic stages of Pseudopterogorgia elisabethae and Pseudopterogorgia bipinnata to biosynthesize biologically active and pharmaceutically important diterpenes was examined. These early life stages lack the algal symbionts found in adult colonies of the species, thus removing one level of complexity. The larvae and polyps of these two...
Show moreThere is growing evidence that secondary metabolites isolated from some marine invertebrates may actually be produced by a symbiont. The capacity of early ontogenetic stages of Pseudopterogorgia elisabethae and Pseudopterogorgia bipinnata to biosynthesize biologically active and pharmaceutically important diterpenes was examined. These early life stages lack the algal symbionts found in adult colonies of the species, thus removing one level of complexity. The larvae and polyps of these two species produced high concentrations of the diterpenoid secondary metabolites, one of which proved to significantly deter fish-feeding in in situ feeding assays. Additionally, a novel cembrenoid diterpene was isolated from P. bipinnata. The structure of this compound, bipinnatolide L (40) was solved using 1D and 2D NMR experiments.
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Date Issued
2005
PURL
http://purl.flvc.org/fcla/dt/13297
Subject Headings
Chemistry, Biochemistry
Format
Document (PDF)
Natural products biosynthesis in the gorgonian Eunicea fusca and other marine invertebrates
Title
Natural products biosynthesis in the gorgonian Eunicea fusca and other marine invertebrates.
Creator
Saleh, Maysoon Baker, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Marine organisms represent a rich source of bioactive secondary metabolites. These bioactive compounds are often present in trace quantities in the organism and their clinical development has been hampered by lack of an available supply. The supply problem can be addressed by a chemical synthesis or through biological methods such as aquaculture, cell culture or enzymatic means. The overall goal of this research was to conduct biosynthetic studies of the marine alkaloids ET 743 and stevensine...
Show moreMarine organisms represent a rich source of bioactive secondary metabolites. These bioactive compounds are often present in trace quantities in the organism and their clinical development has been hampered by lack of an available supply. The supply problem can be addressed by a chemical synthesis or through biological methods such as aquaculture, cell culture or enzymatic means. The overall goal of this research was to conduct biosynthetic studies of the marine alkaloids ET 743 and stevensine, and the diterpenes fuscol and fuscoside A-D. The motivation for this is that biosynthetic data could be used to develop a biotechnological production method. ET 743, an anti-cancer agent, is the main alkaloid produced by Ecteinascidia turbinata. Since DKP of DOPA is a key intermediate in ET 743 biosynthesis and since the chemical synthesis of DOPA derivatives is problematic, we developed an enzymatic production method using tyrosine hydroxylase as a tool to oxidize DKP of tyrosine to DKP of DOPA. Stevensine and oroidin are bromopyrrole alkaloids isolated from the marine sponge Axinella corrugate. Stevensine exhibits antimicrobial activity as well as cytotoxicity in vitro against murine leukemia cells. We were able to develop an enzyme based method to identify the amino acids precursors for Stevensine and oroidin using a cell-free extract of the sponge. Fuscosides are diterpene arabinose glycosides isolated from the Caribbean soft coral Eunicea fusca. They and the aglycone fuscol are potent anti-inflammatory compounds with long-lasting effect and have a selective inhibitory action against leukotriene production in murine models of inflammation. We identified and confirmed the intermediary of the diterpenes cyclase product using radioactivity-guided isolation. Furthermore, we completed the elucidation of fuscol biosynthetic pathway. We provided evidence for the use of these diterpenes as oxygen scavengers in the organism. In addition, we investigated the biosynthetic source of the diterpenes in E. fusca and we provided direct evidence for their production by the microbes (bacteria or fungi) present in the organism.
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Date Issued
2005
PURL
http://purl.flvc.org/fcla/dt/12147
Subject Headings
Chemistry, Biochemistry
Format
Document (PDF)
roles of substrate sequence and thermal stability in the collagenolytic action of matrix metalloproteinases
Title
The roles of substrate sequence and thermal stability in the collagenolytic action of matrix metalloproteinases.
Creator
Minond, Dmitriy, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Matrix metalloproteinases are implicated in diseases accompanied by pathological destruction of tissues. Collagen is an important physiological barrier between tissues and therefore the proteases that can cleave intact collagen are of great interest as possible therapeutic targets. The collagenolytic activity of MMPs varies greatly and within the MMP family distinct preferences for collagen types are seen. Subtle variations in sequence, triple-helix local stability, and Gly register were...
Show moreMatrix metalloproteinases are implicated in diseases accompanied by pathological destruction of tissues. Collagen is an important physiological barrier between tissues and therefore the proteases that can cleave intact collagen are of great interest as possible therapeutic targets. The collagenolytic activity of MMPs varies greatly and within the MMP family distinct preferences for collagen types are seen. Subtle variations in sequence, triple-helix local stability, and Gly register were hypothesized to be among the determinants that may guide these specificities. In order to develop collagenolysis-based inhibitors, we need to examine the interactions between MMPs and collagen on a molecular level. We utilized FRET triple-helical substrates to compare the triple-helical peptidase activities and effects of hypothesized specificity determinants on triple-helical peptidase activities of collagenolytic MMPs. Kinetic parameters of triple-helical peptide hydrolysis were determined for MMP-1, -8, -13, MT1-MMP, and MT2-MMP, and activation energies for this process were calculated. It was determined that MMPs possess differential abilities to process more thermally stable triple-helices. MMP-13 was least sensitive and MMP-1 and MT1-MMP were most sensitive to the enhanced thermal stability of substrates. In agreement with kinetic parameters, hydrolysis of more thermally stable substrates required higher activation energies. MMP collagen specificity also has been examined using a triple-helical substrate library. The consensus type I-III collagen sequence was modified in positions P1' and P2. Single substitution of Gln by Orn in P2 position was beneficial for MMP-13 and MT2 MMP while detrimental for MMP-1, MMP-2, MMP-8, and MT1-MMP activities. Single substitution of Leu by Cys(Mob) in P1' position was disfavored by MMP-1, -2, and -9 and favored by MMP-8, MMP-13, MT1-MMP, and MT2-MMP. Assays of MMP reactions with a sequence containing substitutions in both positions revealed that these sites are not independent from each other, which might indicate an enzyme "induced fit". The significance of triple-helicity for MMP substrate specificity was evaluated by using a library of peptides containing interruptions of Gly register in the vicinity of the scissile bond. Interruption of the Gly register led to the decreased triple-helicity of substrates and subsequently to the inability of membrane type MMPs to cleave such substrates within the interrupted region.
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Date Issued
2006
PURL
http://purl.flvc.org/fcla/dt/12212
Subject Headings
Chemistry, Biochemistry
Format
Document (PDF)
Identification of the diterpene cyclase and elucidation of early steps in the pseudopterosin biosynthetic pathway
Title
Identification of the diterpene cyclase and elucidation of early steps in the pseudopterosin biosynthetic pathway.
Creator
Kohl, Amber Celeste, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
The pseudopterosins and seco-pseudopterosins are diterpene glycosides isolated from the marine soft coral, Pseudopterogorgia elisabethae . These compounds exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. The overall goal of this research was to complete biosynthetic studies and enzymology related to the development of a biotechnological production method of the pseudopterosins and seco-pseudopterosins. We aimed to examine early steps in the...
Show moreThe pseudopterosins and seco-pseudopterosins are diterpene glycosides isolated from the marine soft coral, Pseudopterogorgia elisabethae . These compounds exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. The overall goal of this research was to complete biosynthetic studies and enzymology related to the development of a biotechnological production method of the pseudopterosins and seco-pseudopterosins. We aimed to examine early steps in the biosynthetic pathway in order to gain detailed knowledge of the biosynthesis and enzymology of the system. Prior to examination of the biosynthetic pathway leading to the pseudopterosins, we developed both in vivo and in vitro systems to test putative precursors and demonstrated that pseudopterosin A is a precursor to pseudopterosins B--D. We also identified and confirmed the intermediacy of the pseudopterosin diterpene cyclase product using a radioactivity-guided isolation. The bicyclic structure of the diterpene cyclase product suggests that the pseudopterosins and seco-pseudopterosins are derived from this common bicyclic intermediate. The isolation of the pseudopterosin diterpene cyclase product provided us with an assay for the purification of the enzyme involved in its production. We identified the diterpene cyclase in aqueous extracts of P. elisabethae and plan to utilize the amino acid sequence for identification of the gene. This represents the first isolation of a biosynthetic enzyme from a marine coral. Moreover, various characterization studies indicated that the enzyme displays certain similar characteristics to other terpenoid cyclases isolated from terrestrial sources. In previous investigations, P. elisabethae was found only in the Bahamian and West Indian region, but we discovered P. elisabethae in the Florida Keys. Furthermore, due to the distinctive chemistry in the Florida Keys P. elisabethae, plausible early biosynthetic intermediates were isolated that are not present in the Bahamian population. We evaluated these compounds as intermediates in the biosynthesis of the pseudopterosins. The data obtained further supports the assumption of a common biosynthetic origin of the pseudopterosins and seco-pseudopterosins.
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Date Issued
2003
PURL
http://purl.flvc.org/fcla/dt/12026
Subject Headings
Chemistry, Biochemistry, Chemistry, Organic
Format
Document (PDF)
Regulation of L-glutamic acid decarboxylase by post-translational modifications
Title
Regulation of L-glutamic acid decarboxylase by post-translational modifications.
Creator
Sha, Di, Florida Atlantic University, Charles E. Schmidt College of Science, Center for Complex Systems and Brain Sciences
Abstract/Description
In the central nervous system (CNS), the rate-limiting step in GABA synthesis is the reaction catalyzed by L-glutamic acid decarboxylase (GAD). Alternations in the level of GABA or GAD have been linked to various neurological disorders. Mammalian species express two isoforms of GAD, namely, GAD65 and GAD67, referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Numerous studies have been done to elucidate the mechanisms that control the regulation of GAD at the level of...
Show moreIn the central nervous system (CNS), the rate-limiting step in GABA synthesis is the reaction catalyzed by L-glutamic acid decarboxylase (GAD). Alternations in the level of GABA or GAD have been linked to various neurological disorders. Mammalian species express two isoforms of GAD, namely, GAD65 and GAD67, referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Numerous studies have been done to elucidate the mechanisms that control the regulation of GAD at the level of gene expression, protein synthesis, saturation of co-factor, pyridoxal 5'-phosphate (PLP), and post-translational modification. Our previous studies had demonstrated the presence of the truncated form of human brain L-glutamic decarboxylase 65 (tGAD65) in vivo as well as in vitro and found that tGAD65 was more active than the full-length GAD65 (fGAD65). In addition, the recombinant human brain GAD67 has been found to be specifically cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produce two truncated forms of GAD 67 (tGAD67). It seems that the formation of tGAD is catalyzed by specific proteases instead of a random degradation. Furthermore, it has been found that GAD65 is regulated by the Ca2+-free form of calmodulin (apoCaM). My research focus is to elucidate the regulation of GABA biosynthesis through regulation of its synthesizing enzyme, especially GAD67, by protein phosphorylation, proteolytic cleavage and apoCaM. Experiments presented here have been conducted to demonstrate the molecular cloning, expression, and purification of human brain tGAD67. The purified protein was further characterized by kinetic studies and phosphorylation studies. Truncated forms of hGAD67 were much less active than the full-length form. Both truncated enzymes are also phosphorylated by protein kinase A (PKA) as is full-length hGAD67. A deletion of 1-70 aa from the N-terminal results in additional protein kinase C (PKC) phosphorylation. Several phosphopeptides and possible phosphorylation sites are suggested by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis. Furthermore, evidence of mu-calpain, not m-calpain, as the protease responsible for GAD cleavage in vivo as well as in vitro is presented. In addition, evidence on the effect of ApoCaM on GAD67 activity, phosphporylation and proteolytic cleavage by mu-calpain is discussed. Finally, an overall model of GAD regulation by a variety of mechanisms including protein phosphorylation, mu-calpain proteolytic cleavage and apoCaM is proposed.
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Date Issued
2005
PURL
http://purl.flvc.org/fcla/dt/12170
Subject Headings
Biology, Neuroscience, Chemistry, Biochemistry
Format
Document (PDF)
Bioactive terpene production associated with Caribbean gorgonians from the genera Pseudopterogorgia and Eunicea: Discovery of a sustainable production method
Title
Bioactive terpene production associated with Caribbean gorgonians from the genera Pseudopterogorgia and Eunicea: Discovery of a sustainable production method.
Creator
Newberger, Nealie C., Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Research directed toward renewable, non-destructive sources of bioactive marine derived compounds, is becoming more important everyday. Marine soft corals known as sea whips are a prolific source of such biologically active compounds. One particular organism, Eunicea fusca, possesses diterpene arabinose glycosides with potent antiinflammatory activity that rival the industry standard indomethicin. Fuscocide B is known to inhibit phorbol myristate acetate (PMA)-induced ear edema in murine...
Show moreResearch directed toward renewable, non-destructive sources of bioactive marine derived compounds, is becoming more important everyday. Marine soft corals known as sea whips are a prolific source of such biologically active compounds. One particular organism, Eunicea fusca, possesses diterpene arabinose glycosides with potent antiinflammatory activity that rival the industry standard indomethicin. Fuscocide B is known to inhibit phorbol myristate acetate (PMA)-induced ear edema in murine models, is a minor secondary metabolite and it is therefore difficult to acquire in large quantities. Alternatively, fuscol and eunicol, are known to have similar antiinflammatory activity and are major secondary metabolites which are structurally similar to fuscocide B and are also produced by E. fusca. We have found that fuscol and eunicol can be extracted from the holobiont, and Symbiodinium (Symbiodinium sp.) cells isolated from E. fusca. Similarly, diterpene glycosides, known as pseudopterosins, have been isolated from the the holobiont, and purified Symbiodinium of Pseudopterogorgia elisabethae. Pseudopterosins are also known to possess anti-inflammatory and analgesic properties superior to that of existing drugs. Since many chemically unique metabolites demonstrating bioactive properties have been introduced into pre-clinical and clinical trials I-III5 and the supply issue has been duly highlighted, it has become advantageous to determine the source of these compounds in each of the above mentioned species and determine if a renewable, non-destructive source can be maintained. Data has been presented suggesting that the actual source of the pseudopterosins is not the coral, but actually the dinoflagellate symbiont associated with Pseudopterogorgia elisabethae. Therefore, we have examined cryopreservation of the dinoflagellate symbionts, induction of terpene biosynthesis in the dinoflagellate symbionts, as well as various cell culture techniques in order to better understand the ecological role of terpene biosynthesis in the symbionts and the host corals. The data obtained in the following studies reveals a bacterial source for the production of bioactive secondary metabolites from Eunicea fusca and lends support to a possible bacterial producing trend in gorgonians.
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Date Issued
2006
PURL
http://purl.flvc.org/fcla/dt/12224
Subject Headings
Biology, Oceanography, Chemistry, Biochemistry, Chemistry, Organic
Format
Document (PDF)
Water content, organic content, and carbon and nitrogen composition of medusae from the northeast Pacific
Title
Water content, organic content, and carbon and nitrogen composition of medusae from the northeast Pacific.
Creator
Larson, R. J., Harbor Branch Oceanographic Institute
Date Issued
1986
PURL
http://purl.flvc.org/FCLA/DT/3353781
Subject Headings
Medusae, Biochemistry, Carbon, Nitrogen, Water, Organic and biological chemistry
Format
Document (PDF)
DFT calculations of Amide 1 vibrational frequencies for a model peptide
Title
DFT calculations of Amide 1 vibrational frequencies for a model peptide.
Creator
Lantz, Richard, Stillman, Storm, Terentis, Andrew C.
Date Issued
2012-04-06
PURL
http://purl.flvc.org/fcla/dt/3348894
Subject Headings
Density Functional Theory (DFT), Molecular biology, Amides --chemistry, Biomolecular methods, Biochemistry, Amide-I modes
Format
Document (PDF)