The crystal structures of the pure, unsubstituted firefly emitter oxyluciferin (OxyLH(2)) and its 5-methyl analogue (MOxyLH(2)) were determined for the first time to reveal that both molecules exist as pure trans-enol forms, enol-OxyLH(2) and enol-MOxyLH(2), assembled as head-to-tail hydrogen-bonded dimers. Their steady-state absorption and emission spectra (in solution and in the solid state) and nanosecond time-resolved fluorescence decays (in solution) were recorded and assigned to the six possible trans chemical forms of the emitter and its anions. The spectra of the pure emitter were compared to its bioluminescence and fluorescence spectra when it is complexed with luciferase from the Japanese firefly (Luciola cruciata) and interpreted in terms of the intermolecular interactions based on the structure of the emitter in the luciferase active site. The wavelengths of the emission spectral maxima of the six chemical forms of OxyLH(2) are generally in good agreement with the theoretically predicted energies of the S(0)-S(1) transitions and range from the blue to the red regions, while the respective absorption maxima range from the ultraviolet to the green regions. It was confirmed that both neutral forms, phenol-enol and phenol-keto, are blue emitters, whereas the phenolate-enol form is yellow-green emitter. The phenol-enolate form, which probably only exists as a mixture with other species, and the phenolate-enolate dianion are yellow or orange emitters with close position of their emission bands. The phenolate-keto form always emits in the red region. The concentration ratio of the different chemical species in solutions of OxyLH(2) is determined by several factors which affect the intricate triple chemical equilibrium, most notably the pH, solvent polarity, hydrogen bonding, presence of additional ions, and pi-pi stacking. Due to the stabilization of the enol group of the 4-hydroxythiazole ring by hydrogen bonding to the proximate adenosine monophosphate, which according to the density functional calculations is similar to that due to the dimerization of two enol molecules observed in the crystal, the phenolate ion of the enol tautomer, which is the predominant ground-state species within the narrow pH interval 7.44-8.14 in buffered aqueous solutions, is the most probable emitter of the yellow-green bioluminescence common for most wild-type luciferases. This conclusion is supported by the bioluminescence/fluorescence spectra and the NMR data, as well the crystal structures of OxyLH(2) and MOxyLH(2), where the conjugated acid (phenol) of the emitter exists as pure enol tautomer.

Current models of autosomal recessive polycystic kidney disease (ARPKD) fail to demonstrate biliary abnormalities in association with renal cysts. We therefore studied a new murine model of ARPKD in which dual renal tubular and biliary epithelial abnormalities are present. Affected homozygous animals typically die 1 month postnatally in renal failure with progressively enlarged kidneys. Renal cysts shift in site from inner cortical proximal tubules at birth to collecting tubules 20 days later, as determined by segment-specific lectin binding. Increased numbers of mitosis were demonstrated in proximal and collecting tubular cysts. In addition, epithelial hyperplasia was demonstrated morphometrically in the intra- and extrahepatic biliary tract of affected animals. The number of intrahepatic biliary epithelial cells was increased by 50% on postnatal day 5 and by 100% on postnatal day 25 (P < 0.01). Despite an increased frequency of "chaotic" portal areas in mice with renal cysts, no intrahepatic cysts or shape abnormalities of the biliary lumen were detected using biliary casts and morphometry. Additionally there was nonobstructive hyperplastic dilatation of the extrahepatic biliary tract which was linked in all animals to the presence of renal cysts. The hyperplastic abnormalities in both renal and biliary epithelium make this new mouse strain a good model for the study of the dual organ cellular pathophysiology of ARPKD.

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Spatial microbial distributions of nitrifiers and heterotrophs in undefined mixed‐population biofilms were experimentally investigated using a microslicer technique and correlated with nitrification efficiency of the biofilm system. The general stratification of different bacterial groups in the biofilm was simulated using a one‐dimensional (1‐D) mathematical biofilm accumulation model (BAM) and compared with the experimental results. Biofilms were cultured at three C : N ratios of feed solutions in a partially submerged rotating biological contactor (RBC). It was shown that the biofilms were vertically stratified (from biofilm surface to substratum). At C : N = 0, heterotrophs and nitrifiers coexisted in the outermost biofilm and heterotrophs dominated in the innermost biofilm. At C : N = 1.5, heterotrophs outcompeted nitrifiers for dissolved oxygen and space; thus, heterotrophs dominated in the outermost biofilm and nitrifiers were present only in the deeper biofilm. Nitrifiers and heterotrophs coexisted in the innermost biofilm. An increase in the influent C : N ratio resulted in stronger stratification of microbial species, as well as inhibition of nitrification. In batch experiments, NH4N utilization rate (R NH 4N) was almost the same at each substrate C : N ratio even though NH4 oxidizers were predominantly present in the deeper biofilm. The biofilm performance could not be sufficiently explained by the obtained microbial spatial distribution, suggesting that one‐dimensional description of microbial distribution was not good enough and three‐dimensional measurements of microbial spatial distribution is necessary. Total bacterial densities increased by a factor of 3–17 with biofilm depth. The metabolically active cell fraction decreased from 35 ± 13% in the outermost biofilm to 15 ± 4% in the innermost biofilm, presumably due to substrate limitation. The model predicted more pronounced stratification of nitrifiers and heterotrophs than the observed results. This discrepancy could be attributed to the real biofilms that were structurally heterogeneous (e.g., water channels), which could not be described by the one‐dimensional model. The results of this study clearly indicate the limitation of 1‐D biofilm models to describe the extent of stratification of nitrifiers and heterotrophs and suggest a 3‐D model is necessary. © 1996 John Wiley & Sons, Inc.

Radiation-induced telangiectasia appears to occur in at least 20% of children who undergo cranial irradiation. In this small series, higher radiation dose was not significantly associated with higher frequency of telangiectasia, although there was a trend in this direction.