Studies on the Mechanism of Deoxycytidylate Hydroxymethylase from Bacteriophage T4: A Dissertation

Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-hydroxymethyl-dCMP (Hm5CMP) from dCMP and methylene tetrahydrofolate (CH2THF), analogous to the reaction between dUMP and CH2THF catalyzed by thymidylate synthase (TS), an enzyme of known structure. The amino acid sequence identity between invariant TS residues and CH is at least 50%. Most of the residues which contact the dUMP and CH2THF in TS are conserved in CH. It is hypothesized that CH is homologous to TS in both structure and mechanism. The project described in this thesis tests this hypothesis. In-vitro studies on catalysis by CH variants. The roles of three residues in catalysis by CH have been tested using site-directed mutagenesis. Conversion of Cys148 to Asp, Gly or Ser decreases CH activity at least 105 fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of bound dUMP to the side chain of an Asn; the corresponding CH residue is Asp179. Conversion of Asp179 in CH to Asn reduces kcat/KM for dCMP by 104 fold and increases kcat/KM for dUMP 60 fold, changing the nucleotide specificity of the enzyme. Other studies have shown that the specificity of TS was changed from dUMP to dCMP by conversion of the appropriate Asn to Asp. Based on the crystal structure of TS, a Glu residue (also conserved in CH) is proposed to catalyze formation of the N5 iminium ion methylene donor by protonation of N10 of CH2THF. In CH and TS, overall turnover and tritium exchange are tightly coupled. Replacement of Glu60 in CH or Glu58 in TS uncouples these catalytic steps. Conversion the Glu60/58 to Gln or Asp results in a 5-50 fold decrease in the ability to catalyze tritium exchange, consistent with an inability to catalyze formation of the N5 iminium ion, but also results in a 104-105 decrease in product formation. This suggests that Glu60/58is also involved in a step in catalysis after nucleotide and folate binding and proton removal from carbon 5 of the nucleotide. Isotope effect studies. The observed value of the α-secondary tritium inverse equilibrium isotope effect (EIE = 0.8) on formation of the complex between FdUMP, CH2THF and both wild-type CH and CH(D179N) indicates that carbon 6 of FdUMP is sp3 hybridized (tetrahedral) in the ternary complex. This is consistent with the hypothesis that that carbon 6 is bonded to Cys148 in the complex. Removal of Cys148in CH prevents complex formation with FdUMP. Lack of an observed α-secondary tritium kinetic isotope effect (KIE) for position 6 of dCMP for both enzymes suggests that the intrinsic KIE is masked by other rate-limiting steps or that rehybridization follows the first irreversible step. An observed KIE on carbon 6 of dUMP by CH(D179N) suggests the rate-limiting steps for the two nucleotide substrates is different. In-vivo studies catalysis by CH variants. In order to prevent recombination between CH deficient T4 phage and plasmid borne copies of CH variants, the gene coding for CH, gene 42, was deleted from the T4 chromosome. The T4Δ42 phage requires wild-type CH expressed from a plasmid to kill their host cell. CH variants C148G, D179N, E60Q, and E60D, all which exhibit at least 2000 fold lower activity in vitro, do not complement the T4Δ42 phage in vivo. Interchanging the functional domains of CH and TS. It is proposed that shortening the C-terminal loop seen in the structure of TS changes the solvent structure of the CH active-site such that it becomes more hydrated. Differences in the solvent structure of the active-site may account for differences in the catalytic specificity between CH and TS, respectively, hydration versus reduction. In order to test the hypothesis that these catalytic differences between TS and CH lie within the C-terminal portion of the enzyme, the N-terminus of the CH(D179N) variant was fused to the C-terminus of the wild-type TS to create a chimeric CH/TS enzyme. The chimeric enzyme was predicted to have specificity for dUMP and a active-site solvent structure similar to that for wild-type TS. However, the resulting protein cannot be overproduced to significant levels and does not have any detectable TS activity in vivo

The Impact of the Differentiated Curriculum on African American Women High School Students: The Case in St. Louis

Historians have tagged the transformation to the differentiated curriculum as an influential factor in the shaping of the twentieth-century high school in the United States. Although gender, race, and social class affected the way in which a student\u27s education was altered by the differentiated curriculum, the impact of the differentiated curriculum on African American women high school students has not been studied extensively. This paper examines the effect of the differentiated curriculum on African American women\u27s course selection in St. Louis public high schools from 1914 to 1930. Conclusions drawn here both confirm and challenge the existing literature on women\u27s secondary education

The Needs and Resources of College Women at Morehead State University

A thesis presented to the faculty of the School of Education at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Arts in Adult and Continuing Education by Karen S. Graves in October of 1983

Historias de la Educación LGTBQ+. Introducción

IntroductionIntroduccio

Evaluating the Effectiveness of the Balanced Scorecard Within a Nonprofit Organization

Analyzing and evaluating the effectiveness of nonprofit organizations’ services is as critical as the service itself. The purpose of this study was to identify the effectiveness of the balanced scorecard, a performance measurement system, within a drug and alcohol nonprofit organization and to assess its adaptability for use among nonprofit organizations. The theories used in this study included (a) Kaplan and Norton’s Balanced Scorecard (BSC); (b) Patton’s utilization-focused evaluation; (c) Campbell’s experimental society theory; and (d) Lincoln and Guba’s naturalistic inquiry. The research addressed whether the BSC was a useful evaluation instrument in a nonprofit organization and whether the BSC readily adaptable within its original form. The qualitative research design used was a single case study using both an online survey and conducting an interview with the organization. The responses received by the participating organization were entered into a computerized BSC system and analyzed through a combination of prior case studies, data previously collected from multiple sources including interviews, and a cross-sectional study using the computerized BSC to determine the adaptability of the BSC in a nonprofit environment. The key results suggested the BSC could be an effective evaluation tool in a nonprofit organization; however, based on the limited pool used for this study, the determination that it is readily adaptable in its original form was inconclusive. Findings can be used by organizations to support the use of the BCS, leading to positive social change

Mindfulness-Oriented Recovery Enhancement versus CBT for co-occurring substance dependence, traumatic stress, and psychiatric disorders: Proximal outcomes from a pragmatic randomized trial

In clinical settings, there is a high comorbidity between substance use disorders, psychiatric disorders, and traumatic stress. As such, transdiagnostic therapies are needed to address these co-occurring issues efficiently. The aim of the present study was to conduct a pragmatic randomized controlled trial comparing Mindfulness-Oriented Recovery Enhancement (MORE) to group Cognitive-Behavioral Therapy (CBT) and treatment-as-usual (TAU) for previously homeless men residing in a therapeutic community. Men with co-occurring substance use and psychiatric disorders, as well as extensive trauma histories, were randomly assigned to 10 weeks of group treatment with MORE (n=64), CBT (n=64), or TAU (n=52). Study findings indicated that from pre- to post-treatment MORE was associated with modest yet significantly greater improvements in substance craving, post-traumatic stress, and negative affect than CBT, and significantly greater improvements in post-traumatic stress and positive affect than TAU. A significant indirect effect of MORE on decreasing craving and post-traumatic stress by increasing dispositional mindfulness was observed, suggesting that MORE may target these issues via enhancing mindful awareness in everyday life. This pragmatic trial represents the first head-to-head comparison of MORE against an empirically-supported treatment for co-occurring disorders. Results suggest that MORE, as an integrative therapy designed to bolster self-regulatory capacity, may hold promise as a treatment for intersecting clinical conditions