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Development of Methods for Separation and Characterization of Proteins from Gorgonian Sources
Title
Development of Methods for Separation and Characterization of Proteins from Gorgonian Sources.
Creator
Jett, Steven W., Florida Atlantic University, Kerr, Russell G., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Research into the chemical constituents of marine organisms is a relatively new and growing field, given that much of the marine environment has only in the last half century become accessible. Despite the strides that have been made in ocean exploration, the marine biome has still been only barely explored. Much of the impetus for the isolation and identification of chemical entities from marine organisms has been related to their potential as medicines. For a number of reasons, including...
Show moreResearch into the chemical constituents of marine organisms is a relatively new and growing field, given that much of the marine environment has only in the last half century become accessible. Despite the strides that have been made in ocean exploration, the marine biome has still been only barely explored. Much of the impetus for the isolation and identification of chemical entities from marine organisms has been related to their potential as medicines. For a number of reasons, including synthetic efficiency, this has meant that many marine compounds isolated and studied have been small molecules. In recent years, large strides have been made in techniques for the isolation and identification of biological macromolecules, especially proteins. The understanding of these molecules, and their relationship to the biochemical processes of the marine organisms from which they have been isolated, is important not only because of the potential for this information to help in the synthesis of medicines, but because it may help in the recognition of processes that affect the very viability of marine organisms increasingly exposed to anthropogenic threats to their environment. This work consists of four related studies involving the development of methods for the separation and identification of proteins from a number or gorgon ian species. Chromatographic and gel based methods were used to isolate an elisabethatriene synthase (ELS) which shows promise as a biotechnological tool for the production or pseudopterosins. A number of gorgonians were screened for the presence of proteins that might have antimicrobial properties, and several organisms were identified that might be of interest in this context. Two-dimensional gel electrophoresis methods were then developed to allow the construction of gel maps for the azooxanthellate gorgonian Leptogorgia minimata and the xoozanthellae gorgonian Pseudopterogorgia e/isahethae, the separated proteins were digested and analyzed by LC/MSMS. and the information generated was used to examine the proteome of the organism for functional and phylogenetic relationships. Finally, the gorgonian EuniceafiShow less
Date Issued
2007
PURL
http://purl.flvc.org/fau/fd/FA00000862
Subject Headings
Marine biology, Pharmaceutical biotechnology, Proteins--Analysis, Gene expression--Research--Methodology, Biomolecules--Analysis
Format
Document (PDF)
The Single Minded 2 Gene (SIM2) and Cancer: Harnessing Micro-Array Data to Facilitate Pathway Discovery and Validation
Title
The Single Minded 2 Gene (SIM2) and Cancer: Harnessing Micro-Array Data to Facilitate Pathway Discovery and Validation.
Creator
Aleman, Mireille J., Narayanan, Ramaswamy, Florida Atlantic University
Abstract/Description
A Down's Syndrome related Single Minded 2 gene (SIM2), previously known to be associated with Trisomy 21 was predicted by bioinformatics to be colon cancer specific. In previous work from the laboratory using a patient tissue repository, an isoform of this gene, short form (SIM2-s) was shown to be colon cancer specific. Inhibition of SIM2-s expression by antisense technology resulted in cancer-cell specific apoptosis within 24 hours. Microarray-based gene expression profiling of the antisense...
Show moreA Down's Syndrome related Single Minded 2 gene (SIM2), previously known to be associated with Trisomy 21 was predicted by bioinformatics to be colon cancer specific. In previous work from the laboratory using a patient tissue repository, an isoform of this gene, short form (SIM2-s) was shown to be colon cancer specific. Inhibition of SIM2-s expression by antisense technology resulted in cancer-cell specific apoptosis within 24 hours. Microarray-based gene expression profiling of the antisense-treated colon cancer cells provided a fingerprint of genes involving key cell cycle, apoptosis, DNA damage and differentiation genes. Taking hints from the microarray database, experiments were initiated to decipher the molecular mechanism underlying the cancer specific function of the SIM2-s gene. Using an isogenic cell system, apoptosis was found to be dependent on DNA damage and repair gene, GADD45-a. Further, key pathways including p38 MAP kinase (MAPK) and specific caspases were essential for apoptosis. Programmed cell death was not dependant on cell cycle and was preceded by the induction of terminal differentiation. To clarify whether SIM2-s function is a critical determinant of differentiation, stable transfectants of SIM2-s were established in a murine adipocytic cell line (3T3-L 1 ). SIM2-s overexpression caused a pronounced block of differentiation of the pre-adipocytes into mature adipocytes. A study of the differentiation pathway in 3T3-L 1 cells suggested that this block occurs early on in the cascade. These results supported the starting premise that SIM2-s is a critical mediator of cell differentiation. To clarify whether the SIM2-s gene has transforming potential, the SIM2-s gene was overexpressed in the NIH3T3 murine fibroblast cell line. The cells expressing the human SIM2-s gene exhibited shorter doubling time, abrogation of growth serum requirement, greater cell number at saturation density and focus formation. In vivo tumorigenicity assays showed tumor formation with long latency. These results provide strong evidence for the role of SIM2-s gene in tumor cell growth and differentiation, and validate drug therapy use for the gene.
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Date Issued
2007
PURL
http://purl.flvc.org/fau/fd/FA00000845
Subject Headings
Cancer--Genetic aspects, DNA microarrays--Diagnostic use, Apoptosis--Molecular aspects, Medical informatics, Gene expression--Research--Methodology
Format
Document (PDF)
Molecular pathway identification using microarray technology
Title
Molecular pathway identification using microarray technology.
Creator
Tress, Matthew David., Florida Atlantic University, Narayanan, Ramaswamy
Abstract/Description
Harnessing the human genome using bioinformatics lead to the discovery of a highly cancer-selective gene, Single Minded 2 gene (SIM2). An isoform of the SIM2 gene, the short-form (SIM2-s), was shown to be specific to colon, pancreas, and prostate tumors. Antisense inhibition of SIM2-s in a colon carcinoma derived cell line (RKO) caused inhibition of gene expression, growth inhibition and apoptosis in vitro and in nude mice tumorigenicity models. To understand the mechanism of Sim2-s antisense...
Show moreHarnessing the human genome using bioinformatics lead to the discovery of a highly cancer-selective gene, Single Minded 2 gene (SIM2). An isoform of the SIM2 gene, the short-form (SIM2-s), was shown to be specific to colon, pancreas, and prostate tumors. Antisense inhibition of SIM2-s in a colon carcinoma derived cell line (RKO) caused inhibition of gene expression, growth inhibition and apoptosis in vitro and in nude mice tumorigenicity models. To understand the mechanism of Sim2-s antisense, the antisense treated RKO colon cancer cells were monitored for genome wide expression using Affymetrix GeneChipRTM technology. A list of apoptosis related genes was generated using GeneSpringRTM software. Select GeneChip RTM output was validated by Quantitative RT-PCR. Relevance of a key gene, Growth arrest and DNA damage inducible (GADD45a), in the SIM2-s pathway was established. These results will provide a basis for the future experiments to understand the mechanism underlying Sim2-s activation in specific tumors.
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Date Issued
2004
PURL
http://purl.flvc.org/fcla/dt/13146
Subject Headings
Medical informatics, DNA microarrays--Diagnostic use, Cancer--Genetic aspects, Apoptosis--Molecular aspects, Human genetics--Variation, Gene expression--Research--Methodology
Format
Document (PDF)