Now showing items 1-11 of 11

• #### A study on the interaction of Alzheimer's disease beta amyloid protein with cultured mouse neuroblastoma cell line NB41A3

The $\beta$ amyloid protein is the primary constituent of amyloid plaques in the brains of Alzheimer's disease patients. The generation of $\beta$ amyloid protein from $\beta$ amyloid precursor protein and its interaction with neuronal cells were studied in the mouse neuroblastoma cell line NB41A3. Immunoreactivity to the carboxyl terminal of the precursor protein was detected among the membrane proteins of these cells, indicating that $\beta$ amyloid precursor protein is produced by NB41A3 cells. Also amyloid precursor protein carboxyl terminal immunoreactivity was observed in the conditioned medium of the cells, demonstrating various cytosolic peptide fragments are secreted during the cellular processing of the $\beta$ amyloid precursor protein. Synthetic $\beta$ amyloid peptide was shown to negatively affect NB41A3 neuroblastoma cells as judged by decreasing cell numbers, decreasing amount of cell protein, and release of the cytosolic enzyme, lactic dehydrogenase, into the medium. At the ultrastructural level, internal damage to the nucleus could be observed. Synthetic $\beta$ peptide showed specific binding with neuroblastoma cells. The internalization of the $\beta$ peptide into the cells suggest a direct mechanism for $\beta$ amyloid protein toxicity in vivo. This research contributes to the knowledge of the processing of Alzheimer's disease $\beta$ amyloid precursor protein in NB41A3 cells and demonstrates that NB41A3 cell provides a practical in vitro model for studying the mechanism of Alzheimer's disease and amyloid toxicity.
• #### Benzene and toluene mixing ratios in indoor air of homes with attached garages and measurement of respective biomarkers of exposure and ventilation effects

Benzene and toluene mixing ratios were measured in the indoor air of homes with attached garages for several seasons using a thermal desorption GC-FID sampling and analysis protocol (EPA T0-17). Benzene in the living area of these homes ranged from 1--72 ppbv and toluene ranged 3--111 ppbv. The garage levels of benzene ranged from 8--304 pbbv and the toluene levels ranged from 14--591 ppbv. Numerous experiments and a model support the hypothesis of a single source of toluene and benzene. Source strength estimate calculations supported the hypothesis that gasoline in the attached garage is the primary source of these compounds in living area air. They also showed that the home with the air-to-air heat exchangers and forced ventilation had less transport of aromatics than an unventilated home. Perturbation experiments showed that a metal gas can filled with gasoline in the garage and an indoor window open were important factors for benzene and toluene levels in the living areas of the homes. For most experiments, weighted regression analyses of toluene and benzene mixing ratios were consistent with a sole source. Finally, no correlation was observed between the levels of benzene and toluene measured in living areas and their respective urinary biomarkers: t,t-MA and hippuric acid.
• #### Biochemistry of diatom photosynthetic membranes and pigment-protein complexes

Diatoms are an ecologically important group of algae in both marine and freshwater systems, but in spite of their significance little is known about the structure of their photosynthetic apparatus. This is due in part to the lack of a highly purified, oxygen-evolving thylakoid membrane preparation. Since thylakoid membranes purified from diatoms using methods developed for green plants did not evolve oxygen, a new procedure was developed for use with diatoms. An oxygen-evolving thylakoid membrane preparation is crucial for the study of photosynthetic pigment-protein complexes from these algae because the stability of the Photosystem I (PS I) and Photosystem II reaction centers was shown to be greatly reduced in thylakoid preparations that did not retain electron transport activity. As a result of the instability of PS I in some thylakoid preparations, a novel chlorophyll-binding complex was isolated that contained only the PsaA polypeptide. The isolation of this complex should prove useful in elucidating the structure of the PS I reaction center in all plants. Immunological and N-terminal protein sequencing methods were used to identify several photosynthetic proteins in the purified thylakoid preparation. These results provided evidence for posttranslational modification of two light-harvesting polypeptides (LHCPs) as well as of the PsaB subunit of the PS I reaction center core. Posttranslational modification of LHCPs and/or of PsaB has not been observed in green plants. In contrast to green plants, PS I in diatoms has been shown to be located in the inner thylakoid membranes. It was hypothesized that proteolytic processing of the C-terminus of PsaB in diatoms may be necessary for the PS I holocomplex to be present in the inner membranes, and that this processing may be responsible for the instability of PS I in purified diatom thylakoids. The existence of a functional, highly purified, and extensively characterized thylakoid preparation from diatoms will promote our understanding of the photosynthetic apparatus in these algae.

• #### Structure-Function Studies Of The Serotonin Type -3 Receptor Ligand -Binding Domain

The serotonin type-3 receptor (5-HT3R) is widely distributed in peripheral and central nervous systems. This pentameric protein is a member of the Cys-loop superfamily of ligand gated ion channels and plays a role in mediating physiological processes in nervous, cardiovascular, and digestive systems. The ligand-binding domain of this receptor is extracellularly located and is composed of multiple putative loop structures. Based on structural and sequence homology with other members of the superfamily, it has been proposed that at least six such loops (loops A to F) contribute to the ligand-binding domain. Binding of agonist initiates a conformational change which is transduced to the channel, leading to channel opening (gating). The aim of this study was to elucidate the contribution of residues in loops B and E to the mechanism of channel gating in the 5-HT3R. To this end, the three critical tyrosine residues in the loop E region were characterized employing site-directed mutagenesis, electrophysiological studies as well as radio-ligand binding assays involving two structural classes of 5-HT3R agonists. In addition, structure/function analysis of the loop B region was carried out alanine-scanning mutagenesis. Experimental data were correlated with molecular modeling studies. These studies show that the hydroxytryptamine and phenylbiguanide class of compounds utilize different mechanisms of ligand binding and gating in the 5-HT3R. Studies involving the loop B reveal that this region plays a critical role in ligand binding and channel gating. Data obtained from comparison of ground state and agonist-bound models of the 5-HT 3R was correlated with biochemical data. Taken together, these data suggest that agonist interaction with loop B region probably initiates a conformational wave that results in intra- and inter- subunit hydrogen bonding interactions. Our data suggest that these interactions play a critical role in agonist-induced channel opening.
• #### The lectin-like properties of the extracellular protein produced by Pseudomonas aeruginosa during hexadecane degradation

Numerous microorganisms can degrade hydrocarbons and many produce extracellular compounds. These compounds are generally thought to emulsify hydrocarbons making them more available to the microorganism and stimulating growth. Pseudomonas aeruginosa produces both a rhamnolipid surfactant and an extracellular protein during growth on n-hexadecane (C$\rm\sb{16}H\sb{34}).$ The protein has been hypothesized to stimulate growth by emulsifying hexadecane. However, it has never been shown that the protein has hydrophobic properties characteristic of emulsifiers. An isolation procedure was developed in this study that produces 20-30 mg of very pure protein per 500 ml culture of strain ATCC 17423. The protein is monomeric with a molecular weight of approximately 14,500 determined by SDS-PAGE. Structurally the protein is similar to two other proteins from strains S7B1 and PG210. The proteins from strain S7B1 and 17423 stimulate growth of P. aeruginosa on hexadecane. The hydropathic index of the protein from strain PG201 shows no strong hydrophobic regions in the amino acid sequence. Also the isolated protein from strain 17423 will not bind during hydrophobic chromatography and always acts as a monomer in solution even at concentrations which should cause hydrophobic proteins to aggregate. These results indicate that the protein is not hydrophobic and therefore does not have surfactant-like properties. A surprising result from this investigation is that the protein from strain 17423 has lectin-like (carbohydrate binding) qualities and agglutinates P. aeruginosa, Escherichia coli, human type O, and horse red blood cells. The agglutination is inhibited by EDTA, glucose, mannose and rhamnose. The exact carbohydrate(s) the protein binds has not been determined but evidence suggests that it may bind the carbohydrate portion of the rhamnolipid surfactant. A new model is presented describing the function of the protein. In this model the extracellular protein functions by binding the emulsified hydrocarbon to the outer membrane of the bacterium by both the carbohydrate on the glycolipid surfactant and the lipopolysaccharide of the bacterium. This binding of the hydrocarbon stimulates growth of the bacterium on hexadecane.
• #### Wild-type and mutant 2,2-dialkylglycine decarboxylases: The catalytic role of active site glutamine 52 investigated by site-directed mutagenesis and computer analysis

The ability of pyridoxal 5$\sp\prime$-phosphate (PLP)-dependent 2,2-dialkylglycine decarboxylase (DGD) to catalyze decarboxylation and transamination of amino acids at a single active site depends on the subsite within the active site that cleaves $\alpha$-H and $\alpha$-COO$\sp-$ bonds. As observed in the crystal structure, the strategic position of glutamine 52 at the active site suggests a role in enhancing decarboxylation via formation of a hydrogen bond to the substrate carboxyl group. Supporting evidence for this hypothesis is provided by studies with glutamine 52 active site mutants, computer modeling and protein sequence analyses. Ten mutant DGDs containing alanine, asparagine, aspartate, arginine, glutamate, glycine, histidine, leucine, lysine, and tryptophan at position 52 were produced. All, except the histidine mutant, exhibited decreased rates of decarboxylation compared to wild-type. Histidine and asparagine mutants showed measurable decarboxylation rates. These results and that of wild-type DGD suggest that hydrogen bonding with the substrate is required for decarboxylation. Mutants incapable of hydrogen bonding to the substrate, such as alanine, leucine and tryptophan mutants, showed negligible decarboxylation reactions. Transamination rates increased for some mutants and decreased for others. These data imply that the DGD subsite is influenced by the presence of glutamine 52. Furthermore, there is evidence showing that the subsite environment of wild-type DGD, the histidine and the glutamate mutants are different; the three DGD forms exhibited different chromophores at around $\rm\lambda\sb{max}$ of 500 nm when treated with 2-methylalanine or L-alanine in the presence of 3% glycerol. These results have important implications for other PLP-dependent enzymes, such as ornithine aminotransferase and $\gamma$-aminobutyrate aminotransferase. Since protein sequence alignment indicates DGD is homologous to the two aminotransferases, mutations at amino acid position corresponding to glutamine 52 of DGD at the active sites of these aminotransferases could disrupt the functionality of the enzymes. Protein sequence alignment showed that all but one of the PLP-dependent aminotransferases lack residues at position 52 capable of hydrogen bonding with the substrate carboxyl group, further re-affirming the role of glutamine 52 in decarboxylation.