UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Policy analysis of the Americans with Disabilities Act of 1990 and federal funding concerning barrier-free playgrounds

The Americans with Disabilities Act (ADA) of 1990 was created to prohibit discrimination against disabled persons in our society. The goal of the ADA as a comprehensive civil rights law is to "ensure equal opportunity and complete participation, independent living and economic self-sufficiency" for disabled persons (U.S. Department of Justice, 2008). As part of Title II and III of the ADA, states and local governments are required to provide people with disabilities the same chance to engage in and benefit from all programs and services including recreational facilities and activities as every other citizen. Recreational facilities and related structures must comply with accessibility standards when creating new structures or renovating existing ones. Through a systematic literature review of articles accessed through online databases, articles relating to children with disabilities, their quality of life and their experience gained through play were reviewed, analyzed and synthesized. Additionally, the ADA's Final Rule regarding accessible playgrounds was evaluated through a descriptive analysis which yielded the following five components relating the importance of barrier-free playgrounds to children with disabilities: appropriate dimensions for children, integration of the play area, variety of activity and stimulation, availability of accessible play structures to communities, and financial feasibility. These components were used as evaluation criteria to investigate the degree to which the ADA's Final Rule document met these criteria. An evaluation of two federal funding sources, the Urban Parks and Recreation Renewal Program (UPARR) and the Land and Water Conservation Fund (LWCF), was also conducted which revealed three components relating the two programs' ability to support the realization of the ADA's Final Rule which included: current budget for the program, ability of local communities to attain funds, and level of ADA compliance required to receive funding. Majority of the evaluation of the Final Rule concluded it be adequate in development of barrier-free playgrounds although there are some portions of the guidelines that would benefit from further elucidation. Both funding programs were concluded to not adequately support the development of barrier-free playgrounds and therefore it was recommended that their funding be re-instated or increased as necessary. ^

The Ryan White CARE Act : 20 years of transition

The Ryan White CARE Act has undergone substantial changes in the past 20 years. Today it serves over 500,000 persons living with HIV/AIDS in the United States. The purpose of this project was to update the history and content Ryan White CARE Act and to consider how changes since the 2006 CARE Act reauthorization have affected the Ryan White Planning Council in Harris County. The results of the 2008 Houston Area HIV/AIDS Needs Assessment were reviewed and compared to responses to access of service questions of the 2005 Houston Area HIV/AIDS Needs Assessment. The results of the comparison show that informational barriers continue to be the leading barriers in persons living with HIV/AIDS to accessing services. In conclusion the strength of the Ryan White CARE Act is its ability to respond to a changing epidemic and its weakness lies in its vulnerability to shifting political sentiments. ^