The crystal structures of family 10 xylanases indicate that the distal regions of their active sites are quite different, suggesting that the topology of the substrate binding clefts of these enzymes may vary. To test this hypothesis, we have investigated the rate and pattern of xylooligosaccharide cleavage by the family 10 enzymes, Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) and Cellulomonas fimi exoglucanase, Cex. The data showed that Cex contained three glycone and two aglycone binding sites, while XYLA had three glycone and four aglycone binding sites, supporting the view that the topologies of substrate binding clefts in family 10 glycanases are not highly conserved. The importance of residues in the substrate binding cleft of XYLA in catalysis and ligand binding were evaluated using sitedirected mutagenesis. In addition to providing insight into the function of residues in the glycone region of the active site, the data showed that the aromatic residues Phe-181, Tyr-255, and Tyr-220 play important roles in binding xylose moieties, via hydrophobic interactions, at subsites ؉1, ؉3, and ؉4, respectively. Interestingly, the F181A mutation caused a much larger reduction in the activity of the enzyme against xylooligosaccharides compared with xylan. These data, in conjunction with a previous study (Charnock, S. J., Lakey, J. H., Virden, R., Hughes, N., Sinnott, M. L., Hazlewood, G. P., Pickersgill, R., and Gilbert, H. J. (1997) J. Biol. Chem. 272, 2942-2951), suggest that the binding of xylooligosaccharides at the ؊2 and ؉1 subsites ensures that the substrates occupy the ؊1 and ؉1 subsites and thus preferentially form productive complexes with the enzyme. Loss of ligand binding at either subsite results in small substrates forming nonproductive complexes with XYLA by binding to distal regions of the substrate binding cleft.
A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17β-estradiol (E 2 ). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology-driven clustering, was used to define the transcriptional program associated with E 2 -induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data.
ArticlesIn this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the uterotrophic activity of 4MBC.
Two important factors that determine the flux of hepatic beta-oxidation of long-chain fatty acids are the availability of fatty acid and the activity of carnitine palmitoyltransferase I (CPT I). Using Metabolic Control Analysis, the flux control coefficient of CPT I in rat hepatocyte monolayers was determined by titration with 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (Etomoxir), which is converted to Etomoxir-CoA, an irreversible inhibitor of CPT I. We measured CPT I activity and flux through beta-oxidation at 0.2 mM and 1.0 mM palmitate to simulate substrate concentrations in fed and fasted states. Rates of beta-oxidation were 4.5-fold higher at 1. 0 mM palmitate compared with 0.2 mM palmitate. Flux control coefficients of CPT I, estimated by two independent methods, were similar: 0.67 and 0.79 for 0.2 mM palmitate, and 0.68 and 0.77 for 1 mM palmitate. It is concluded that the regulatory potential of CPT I is similar at low and high physiological concentrations of palmitate.
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