A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.

Mice of the inbred strain DBA/2 responded to a two-stage, initiation-promotion tumorigenesis protocol when high initiating doses (400 nmol/mouse) of 7,12-dimethylbenz[a]anthracene were utilized. They also responded when N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as the initiating agent. The tumor response in both cases was characterized by a rapid rate of tumor development with the maximal tumor responses reached on or before the 15th week of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). When DBA/2 mice were compared with SENCAR mice for promotion sensitivity following initiation with MNNG, the two mouse stocks responded with a nearly identical tumor response. C57BL/6 mice were essentially resistant to TPA promotion regardless of the initiator or the dose of initiator used. A preliminary study was conducted to determine how susceptibility to tumor promotion by TPA was inherited in F1 mice derived from DBA/2 (sensitive) and C57BL/6 (resistant) parents. The B6D2F1 mice were as sensitive as the DBA/2 parent, suggesting that susceptibility in these two inbred mouse strains is inherited as an autosomal dominant trait. The results show that these two inbred mouse strains may provide a model system for studying genetic factors controlling susceptibility to phorbol ester skin tumor promotion.

Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.

The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.