Characteristics of multicellular spheroids formed by primary cultures of human thyroid tumor cells

Features of multicellular tumor spheroids (MCTS) differed depending on their types of cells. MCTS formed by 4000 human thyroid primary culture epithelial tumor cells displayed diameters between 0.31 and 0.33 mm within 2 days regardless of the stage of malignancy of the originating tumors. Their cellular composition reflected that of the originating tumor in regard to DNA content and the expression of cytokeratin, vimentin, as well as thyroglobulin. During the following 3 weeks, their sizes increased up to diameters of 0.42 mm when their cells had been derived from carcinomas, and MCTS originating from adenomas stopped growing within the next 2 days. After 8 days of incubation, proliferating cells were only found in carcinoma MCTS. The cells were randomly distributed over the total volume of the spheroids, which displayed irregular cell arrangements but not concentric cell layers and did not form necrotic centers.     

Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase

Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.     

Data sources for pharmacoeconomic and health services research

Different types of databases available for health-related research, the data contained in these databases, and potential applications for pharmacists or researchers are discussed. Case studies that demonstrate uses for health databases are presented. Databases can be organized by facility, by health care provider, by disease or organ, or by sector. The types of data they contain include financial data, utilization data, demographic data, and outcomes data. Data can be obtained from the public sector, the private sector, or the researcher's own health system. The costs and time associated with using existing databases are often less than those required to collect data, but the quality and accessibility of the data must also be considered. The researcher's choice of database will depend on the research question. Health care databases can be used for health management and decision-making, quality review and evaluation, outcomes research, episode-of-illness studies, and evaluation of treatment protocols. Researchers must comply with patient-confidentiality and other agreements when accessing data. The format of the data needs to be matched with the hardware and software to be used in the analysis, and the data need to be loaded, verified, and cleaned before use. In deciding which of the many available data sources to use, researchers must determine the appropriate balance between external data and data available within their own health systems. The decision on whether to use existing data sources or to collect data prospectively will depend on the research question, the available resources, and the scope of the study.     

Nitroreduction of 2,4-dinitrotoluene in vitro by cytochrome P-450 induced H4IIE cells

Conditions have been established for H4IIE rat hepatoma cell cultures in which effects of cytochrome P-450 induction on the metabolism of a munitions wastestream pollutant can be studied. Under these conditions, the polychlorinated hydrocarbon 2,3,4,7,8-pentachlorodibenzfuran (PCDBF) induced cytochrome P-450 (1A1) aryl hydrocarbon hydroxylase (AHH) activity over a wide range of concentrations without significant cytotoxic effects. The munition pollutant 2,4-dinitrotoluene (2,4-DNT) did not induce AHH activity itself, but its metabolism was considerably altered when applied to PCDBF induced cultures. Production of amino nitrotoluene isomers was greatly enhanced in induced cultures as compared to uninduced controls, as was the conversion of radiolabeled 2,4-DNT to relatively more polar metabolites. To some extent, the results with H4IIE cells parallel those reported for animals exposed to 2,4-DNT after induction of cytochrome P-450 AHH activity. The preliminary findings suggest that with further development and validation, H4IIE cultures could be of use in characterizing metabolites that result from exposure to chemical mixtures involving a P-450 (1A1) inducer.     

New genetic loci that control susceptibility and symptoms of experimental allergic encephalomyelitis in inbred mice

Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis, is a genetically determined phenotype. In this study, analyses of the cumulative disease frequencies in parental, F1 hybrid, and F2 mice, derived from the EAE-susceptible SJL/J strain and the EAE-resistant B10.S/DvTe strain, confirmed that susceptibility to EAE is not inherited as a simple Mendelian trait. Whole genome scanning, using 150 informative microsatellite markers and a panel of 291 affected and 390 unaffected F2 progeny, revealed significant linkage of EAE susceptibility to marker loci on chromosomes 7 (eae4) and 17, distal to H2 (eae5). Quantitative trait loci for EAE severity, duration, and onset were identified on chromosomes 11 (eae6, and eae7), 2 (eae8), 9 (eae9), and 3 (eae10). While each locus reported in this study is important in susceptibility or disease course, interactions between marker loci were not statistically significant in models of genetic control. One locus, eae7, colocalizes to the same region of chromosome II as Orch3 and Idd4, susceptibility loci in autoimmune orchitis and insulin-dependent diabetes mellitus, respectively. Importantly, eae5 and eae7 are syntenic with human chromosomes 6p21 and 17q22, respectively, two regions of potential significance recently identified in human multiple sclerosis genome scans.