Pharmaceutical biotechnology

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 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 Euniceafi<sca was exposed to several environmental stressors in
the laboratory. and the two-dimensional proteomic methods developed were used to
examine the effect of the stressors on the organism .

The effect of processing temperature on the crystal structure properties of the Fe-substituted Hydroxyapatite (Fe-HAp) was studied by using the Rietveld refinement method of powder x-ray (XRD) and neutron diffraction (NPD) patterns. Superconducting QUantum Interference Device (SQUID) magnetometry, transmission electron microscopy (TEM) and x-ray fluorescence spectroscopy (XRF) were used to study the magnetic properties, particle morphology and chemical composition of the prepared samples. Two sets of samples of chemical formula Ca5-xFex(PO4)3OH were prepared with x = 0, 0.05, 0.1, 0.2 and 0.3 by using processing temperatures of 37°C and 80°C, following a two-step co-precipitation method. A single phase HAp was identified in samples with x = 0 and 0.05. Processing temperature affects the type and percentage of secondary phases: hematite was detected in samples prepared at 37°C with x ≥ 0.1, hematite and maghemite were detected in samples prepared at 80°C with x = 0.2 and 0.3. Rietveld refinements of NPD and XRD patterns showed that the a lattice constants are greater in Fe-substituted samples prepared at 37°C, whereas the c lattice constants are greater in the 80°C samples for x ≥ 0.05. Fe preferentially substitutes at the Ca2 site in the 80°C samples, whereas Ca1 is the preferred substitution site in the 37°C samples. Fe substitution results to a decrease of the lattice constants at both preparation temperatures. The ratios Fe/(Fe + Ca) of the refined atomic fractions of the samples prepared at 80°C are greater than those of the 37°C samples. Further, more secondary phases form in samples prepared at 37°C compared to 80°C samples. The magnetic measurements reveal that pure HAp is diamagnetic, whereas samples with x = 0.05 and 0.1 are paramagnetic. Samples with x = 0.3 showed superparamagnetic behavior based on ZFC and FC measurements. Similar hysteresis loops in samples x = 0.2 and 0.3 indicate that the samples with x = 0.2 may show superparamagnetic properties. For x = 0.2 and 0.3, the samples prepared at 80°C showed higher magnetization compared to the 37°C samples, because of the maghemite secondary phase. Based on the TEM images, Fe substituted HAp nanoparticles prepared at 37°C are mainly spherically shaped, and the 80°C particles are mainly elongated. Increase of the Fe concentration favors formation of elongated particles and larger spherical particles. The XRF measurements confirm the Fe for Ca substitution in the HAp structure based on the decrease of the Ca/P and the increase of the Fe/(Fe + Ca) atomic ratios with the Fe concentration.

Carbohydrate recognition is one of the most sophisticated recognition processes in biological
systems, mediating many important aspects of cell-cell recognition, such as inflammation, cell
differentiation, and metastasis. Consequently, lectin-glycan interactions have been intensively
studied in order to mimic their actions for potential bioanalytical and biomedical applications.
Galectins, a class of ß-galactoside-specific animal lectins, have been strongly implicated in
inflammation and cancer. Galectin-3 is involved in carbohydrate-mediated metastatic cell
heterotypic and homotypic adhesion via interaction with Thomsen-Friedenreich (TF) antigen on
cancer-associated MUC1. However, the precise mechanism by which galectin-3 recognizes TF
antigen is poorly understood. Our thermodynamic studies have shown that the presentation of the
carbohydrate ligand by MUC1-based peptide scaffolds can have a major impact on recognition,
and may facilitate the design of more potent and specific galectin-3 inhibitors that can be used as
novel chemical tools in dissecting the precise role of galectin-3 in cancer and inflammatory
diseases. Another lectin, odorranalectin (OL), has been recently identified from Odorrana grahami
skin secretions as the smallest cyclic peptide lectin, has a particular selectivity for L-fucose and
very low toxicity and immunogenicity, rendering OL an excellent candidate for drug delivery to
targeted sites, such as: (1) tumor-associated fucosylated antigens implicated in the pathogenesis
of several cancers, for overcoming the nonspecificity of most anticancer agents; (2) the olfactory epithelium of nasal mucosa for enhanced delivery of peptide-based drugs to the brain.