Inducible cytochrome P450 activities in renal glomerular mesangial cells: biochemical basis for antagonistic interactions among nephrocarcinogenic polycyclic aromatic hydrocarbons

BACKGROUND: Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes in vitro and in vivo. While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons. METHODS: To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 - 30 microM) or in the presence of ANTH (3 microM) or CHRY (3 microM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC). RESULTS: Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism. CONCLUSION: These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential.     

NMR structure of stem-loop D from human rhinovirus-14

The 5'-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem-loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3Cpro. This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3Dpol) for replication. Human rhinovirus-14 (HRV-14) is distinct from the majority of picornaviruses in that its SLD forms a cUAUg triloop instead of the more common uYACGg tetraloop. This difference appears to be functionally significant, as 3Cpro from tetraloop-containing viruses cannot bind the HRV-14 SLD. We have determined the solution structure of the HRV-14 SLD using NMR spectroscopy. The structure is predominantly an A-form helix, but with a central pyrimidine-pyrimidine base-paired region and a significantly widened major groove. The stabilizing hydrogen bonding present in the uYACGg tetraloop was not found in the cUAUg triloop. However, the triloop uses different structural elements to present a largely similar surface: sequence and underlying architecture are not conserved, but key aspects of the surface structure are. Important structural differences do exist, though, and may account for the observed cross-isotype binding specificities between 3Cpro and SLD.     

Mycophagy by small mammals in the coniferous forests of North America: nutritional value of sporocarps of Rhizopogon vinicolor, a common hypogeous fungus

We evaluated the nutritional value of sporocarps of Rhizopogon vinicolor, a common hypogeous fungus in the coniferous forests of North America, for two small mammal species: the Californian red-backed vole (Clethrionomys californicus) and the northern flying squirrel (Glaucomys sabrinus). Although the nitrogen concentration of sporocarps was high, much of it was in non-protein form or associated with cell walls, suggesting that it may be of low nutritional value or protected from mammalian digestive enzymes. Sporocarps also had high concentrations of cell wall constituents, indicating low availability of digestible energy. When fed a diet of this fungus alone in a controlled feeding experiment both mammal species lost a small amount of body mass. Digestibilities of dry matter, nitrogen, cell wall constituents and energy from sporocarps by both species were lower than the digestibilities of other food types by other similarly sized small mammals. Red-backed voles digested the various components of sporocarps at least as well as the flying squirrels, even though they were almost six-fold smaller in body mass. This observation supports the notion that red-backed voles, like other microtine rodents, have morphological and physiological adaptations of the digestive system that are postulated to permit greater digestion of fibrous diets than predicted on the basis of body size. Despite this, our results re-affirm previous conclusions that hypogeous fungi are only of moderate nutritional value for most small, hindgut-fermenting mammals. Future studies should focus on the importance of mixed-species of fungi in the diet of small mammalian mycophagists. Accepted: 4 December 1998     

Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyzes formation of the β-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O₂)-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O₂ to the IPNS–Fe(II)–ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed ¹⁹F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O₂ analog nitric oxide, using the less mobile α6 for comparison. ¹⁹F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron–electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.