Biomedical subjects
Publications and source records attributed to F W Frueh.
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Explore the source record for details and available documents.
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This work describes the in situ synthesis of oligonucleotide arrays on glass surfaces. These arrays are composed of features defined and separated by differential surface tension (surface tension arrays). Specifically, photolithographic methods were used to create a series of spatially addressable, circular features containing an amino-terminated organosilane coupled to the glass through a siloxane linkage. Each feature is bounded by a perfluorosilanated surface. The differences in surface energies between the features and surrounding zones allow for chemical reactions to be readily localized within a defined site. The aminosilanation process was analyzed using contact angle, X-ray photoelectron spectroscopy (XPS), and time-of-flight/secondary ion mass spectroscopy (TOF-SIMS). The efficiency of phosphoramidite-based oligonucleotide synthesis on these surface tension arrays was measured by two methods. One method, termed step-yields-by-hybridization, indicates an average synthesis efficiency for all four (A,G,C,T) bases of 99.9 +/- 1.1%. Step yields measured for the individual amidite bases showed efficiencies of 98.8% (dT), 98.0% (dA), 97.0% (dC), and 97.6% (dG). The second method for determining the amidite coupling efficiencies was by capillary electrophoresis (CE) analysis. Homopolymers of dT (40- and 60mer), dA (40mer), and dC (40mer) were synthesized on an NH(4)OH labile linkage. After cleavage, the products were analyzed by CE. Synthesis efficiencies were calculated by comparison of the full-length product peak with the failure peaks. The calculated coupling efficiencies were 98.8% (dT), 96.8% (dA), and 96.7% (dC).
One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5 x 7.5 cm) covering a surface area of 2.25 cm(2). We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.
Phenobarbital elicits pleiotropic effects in the liver, including induction of enzymes involved in xenobiotic metabolism. The spectrum of this response was analyzed by differential display of a large population (approximately 7500) of mRNAs in chicken embryo liver treated in vivo with phenobarbital. We identified 29 cDNA fragments that reproducibly and significantly changed in intensity after a 48-hr in ovo treatment. Eighteen of these (62%) were increased, whereas 11 (38%) were decreased. Twenty strongly regulated cDNA fragments were subcloned and further analyzed. Nucleotide sequence analysis revealed three types of genes: (a) those previously described to be regulated by phenobarbital, including CYP2H1, glutathione S-transferase, and uridine diphosphate-glucuronosyltransferase; (b) genes reported herein for the first time to be regulated by phenobarbital, including fibrinogen beta-chain and gamma-chain, retinal glutamine synthetase, apolipoprotein B, two gene products with homologies to elongation factor 1delta and complement factor H, respectively, and (c) several novel genes with hitherto unknown functions. If these data are extrapolated to the entire population of mRNAs of a liver cell, phenobarbital seems to significantly modulate the expression of more than 50 different genes. Our results also demonstrate that a large fraction of genes is negatively regulated by drug treatment.