Hox proteins are evolutionarily conserved transcription factors that control important developmental pathways in morphogenesis of the embryo. The Hoxa2 gene is expressed in the developing central nervous system in rhombomeres 2 to 7 and affects cellular differentiation. Few target genes of Hoxa2 protein have been identified so far and its mechanisms of regulating gene expression remain elusive. Previous work in our laboratory isolated Hoxa2 protein binding sequences from the E18 mouse spinal cord and hindbrain tissues using chromatin immunoprecipitation (ChIP). All isolated DNA fragments contain conserved GATG motifs. Sequence analysis revealed that one fragment belongs to the high temperature requirement factor A 3 (HtrA3) gene and another fragment belongs to the Dual specificity tyrosine kinase 4 (Dyrk4) gene. In this study, direct binding of Hoxa2 protein to the HtrA3 and Dyrk4 fragments was confirmed by electrophoretic mobility shift assays (EMSA). Site-directed mutagenesis and EMSA studies revealed that Hoxa2 protein binds to the multiple GATG motifs within these fragments. HtrA3 fragment also repressed luciferase gene expression in transient transfection and luciferase assays. Mutation of the DNA fragment showed that the repressive activity was affected by the GATG motifs, suggesting Hoxa2 protein regulated gene expression by binding to the GATG motif in the cis-regulatory element. In contrast to the inhibitory activity of Hoxa2 protein, a Hoxa2-VP16 fusion protein (Hoxa2 fused with an activation domain of a virion protein from herpes simplex virus) transactivates the luciferase expression by binding to GATG sites. RT-PCR and immunohistochemistry analysis revealed an upregulation of HtrA3 expression in Hoxa2-/- mice. This observation correlates with the inhibitory role of Hoxa2 protein acting upon the HtrA3 fragment in luciferase assays. Our data suggest that HtrA3 is a direct in vivo downstream target of Hoxa2 protein and support the activity regulation model in which Hox proteins selectively regulate target genes through occupation of multiple monomer binding sites.

The extracellular polymeric substances (EPS) of marine bacterium Sagittula stellata and soil bacterium Pseudomonas fluorescens Biovar II, were extracted by six methods referred to the bibliography, efficacies of which were compared based on the EPS yield, composition as well as cell disturbance. Purification methods on these EPS were also improved, which proved to be more cost-effective and involve less interference from broth, compared to previous methods. Size exclusion chromatography (SEC) proved to be a useful tool, providing the “fingerprints” of the EPS extracted by different methods or after each purification step. Studies of the EPS production and composition at different growth stages provided abundant information and a basis for further in-depth studies. Results from SEC demonstrated that bacterial EPS had a constant molecular weight distribution all through the life but with various polymers in different proportions. Three fractions were successfully isolated by a combination of SEC and anion exchange chromatography for “non-attched” EPS produced by Pseudomonas flurorescens Biovar II. Protein turned out to be a major component of EPS in their native states, which was mixed with the broth material and couldn’t be recognized previously. The EPS harvested at the optimal time of the bacterial life was purified according to the improved method and was more enriched in polysaccharides, with small amounts of proteins, giving the molecules amphiphilic properties. In addition, simultaneous determination of neutral sugars and uronic acids by GC-EI-MS provided more information on the monosaccharide composition of the exopolysaccharides. Isoelectric focusing (IEF) spectra of the bacterial EPS spiked with Pu/Th, and Pu-enriched Rocky Flats Environmental Technology Site (RFETS) soil organic colloid spiked with Th showed similar activity distributions of both actinides along the pH gradient, with the activities of both actinides focusing on the low pH region. Characterizations of this Pu-enriched IEF extract from RFETS soil by spectrophotometric methods and ATR-FTIR indicated the co-presence of lipids, proteins and polysaccharides, in contrast to the bacterial EPS, which showed a simpler composition. This suggests that Th/Pu binding to organic macromolecules is more determined by the availability of binding functional groups rather than the exact specific compounds.

Polyamide containing groove binding agents have been widely reported We now synthesized netropsin analogs containing azo¡Bimine and diimine functional groups in place of the amide functionality and study their biological activities. Some of these analogs were found to have cytotoxicity in the range of 5 g/mL.

Polyamide containing groove binding agents have been widely reported We now synthesized netropsin analogs containing urea groups in place of the amide functionality and study their biological activities.

Polyamide containing groove binding agents have been widely reported. We synthesized pyrrolidine to displace one of pyrrole of distamycin, and research into structure which was effected by optical and saturated heterocycle.

Polycyclic aromatic hydrocarbons (PAHs) are in general characterized by high toxicity, persistance, and bio-accumulation in environment. It is important to understand the transport and fate of PAHs in environment. In aquatic environment, PAHs are easily bound with dissolved organic matter (DOM), such as humic substance, and KDOC is usually used in denoting the binding constant. Previously, many investigations focused on the binding of a single PAH to DOM. However, few researches considered the competition effect on the binding of multiple PAHs to DOM. This study therefore explored the competition between the binary PAHs, pyrene and phenanthrene, in binding with humic substances. No significant competition effect in KDOC could be observed between these two compounds with LHA (Leonardite Humic Acid) because of the relatively large variation in KDOC determination. Humic substances from different sources might have different properties. Several characteristic indicators of humic substances were used to interprete the variations of KDOC of pyrene and phenanthrene, such as elemental ratio, total acidity, functional group content, etc. Negative correlations of O/C and phenolic group content to the KDOC of pyrene and phenanthrene were observed (p<0.05). In contrast, positive correlation of aromaticity and £`280 of humic substance to the KDOC of pyrene and phenanthrene were observed (p<0.05). Therefore, the contents of phenolic and aromatic groups of humic substance are essential factors in determining their binding constants with PAHs.

Aromatic oligoamide foldamers has high potential to mimic the secondary structures of biopolymers. These oligomers by using intramolecular hydrogen bonds and £k-£k interaction of aromatic rings to form a stable foldamer. We use the helical form of these oligomers and combine to the groove binding agents, then study the influence on DNA .

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, December 2003. / Includes bibliographical references. Also available on the Internet.

A series of conformationally constrained and flexible pseudopeptides were prepared and their thermodynamic parameters on binding to the Grb2 SH2 domain were determined by isothermal titration calorimetry (ITC). Cyclopropane constrained analogs having hydrophobic amino acid residues at the pTyr+1 position exhibited, on average, a 2-5 fold improvement in binding affinities with the enhancement in affinities due to a more favorable enthalpy, not entropy, of binding. This serves as the first set of examples which demonstrate that favorable entropies of binding are not an inherent characteristic of ligand preorganization. Incorporation of polar amino acid residues at the pTyr+1 position eventuated in a slightly different thermodynamic effect than what was observed with the hydrophobic analogs. The constrained molecules exhibited greater binding affinities for the Grb2 SH2 domain and that increase in affinity was a consequence of a more favorable enthalpy, not entropy, of binding. However, the binding entropies for the polar set of constrained and flexible molecules were all negative. Structural information obtained from the co-crystallization of selected constrained and flexible ligand pairs with the Grb2 SH2 domain revealed an increase in conformational mobility of the BC loop in the complexes of the constrained derivatives and the presence of a greater number of direct polar contacts, but fewer water-mediated interactions between the phosphate group of the constrained molecules and the pTyr binding pocket of the domain. The construction of the cis-hydrindanone ring system in sieboldine A was accomplished utilizing either a Lewis acid-mediated silyl-directed Nazarov cyclization of a functionalized divinyl ketone or a sequential Ni(0)-catalyzed 1,4-addition/5-endo-dig cyclization. Propargylzinc bromide was shown to undergo conjugate addition to the [alpha]-aminopropyl substituted enone using Ni(acac)₂, thus providing a new, mild protocol for the conjugate propargylation reaction. Further efforts toward the formation of the α-epoxy ketone are described. / text

Not available / text