PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^

Locus of control as a predictor of students dropping out of high school

Education is related to health. In cross-sectional data, education level has been associated with physical functioning. Also, lower levels of education have been associated with health behaviors including smoking, alcohol use, and greater body weight. In school, students may benefit from greater exposed to health-related messages, while students who have dropped out may be more susceptible to influences regarding negative health behaviors such as smoking. ^ Improved school retention might improve long-term health outcomes. However, there is limited evidence regarding modifiable factors that predict likelihood of dropping out. Two likely psychosocial measures are locus of control and parent-child academic conversations. In the current study, data from two waves of a population-based longitudinal survey, the National Education Longitudinal Survey, were utilized to evaluate whether these two psychosocial measures could predict likelihood of dropping out, for students (n = 16,749) in tenth grade at 1990, with dropout status determined at 1992, while controlling for recognized sociodemographic predictors including parental income, parental education level, race/ethnicity, and sex. Locus of control was measured with the Pearlin Mastery Scale, and parent-child academic conversations were measured by three questions concerning course selection at school, school activities and events, and things the student studied in class. ^ In a logistic regression model, with the sociodemographic control measures entered in a first step before entry of the psychosocial measures in a second step, this study determined that lower levels of locus of control were associated with greater likelihood of dropping out after two years (odds ratio (OR) = 1.11, 95% confidence interval (CI) 108 to 1.15, p < .001), and two of the three parent-child academic discussion items were associated with greater likelihood of dropping out after two years (OR = 1.69, CI 1.48-1.93, p < .001; OR = 1.22, CI 1.05-1.41, p = .01; OR = 1.01, CI .88-1.15, p = .94). ^ It is possible that interventions aimed at improving locus of control, and aimed at building parent-child academic conversations, could lower the likelihood of students dropping out, and this in turn could yield improved heath behaviors and health status in the child's future. ^