Combined histomorphometric and gene-expression profiling applied to toxicology

We have developed a unique methodology for the combined analysis of histomorphometric and gene-expression profiles amenable to intensive data mining and multisample comparison for a comprehensive approach to toxicology. This hybrid technology, termed extensible morphometric relational gene-expression analysis (EMeRGE), is applied in a toxicological study of time-varied vehicle- and carbon-tetrachloride (CCl₄)-treated rats, and demonstrates correlations between specific genes and tissue structures that can augment interpretation of biological observations and diagnosis.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Technicolour transgenics: imaging tools for functional genomics in the mouse

Over the past decade, a battery of powerful tools that encompass forward and reverse genetic approaches have been developed to dissect the molecular and cellular processes that regulate development and disease. The advent of genetically-encoded fluorescent proteins that are expressed in wild type and mutant mice, together with advances in imaging technology, make it possible to study these biological processes in many dimensions. Importantly, these technologies allow direct visual access to complex events as they happen in their native environment, which provides greater insights into mammalian biology than ever before.     

"Delayed" phase separation in a gelatin/dextran mixture studied by small-angle light scattering,turbidity, confocal laser scanning microscopy,and polarimetry

Small-angle light scattering, turbidity, and confocal laser scanning microscopy were used to study microstructure formation and evolution in a gelatin/dextran mixture. There was a time-delay of up to tens of minutes between reaching the quench temperature and the onset of phase separation, because demixing only occurred once a certain amount of ordering of the gelatin molecules, measured by polarimetry, was attained. The accompanying phenomenon of gelation retarded the development of the microstructure to different extents, depending on the quench temperature. At low temperatures, the structure was rapidly trapped in a nonequilibrium state with diffuse interfaces, characteristic of the early and intermediate stages of phase separation. At higher temperatures, coarsening continued for a certain amount of time before the structure was trapped. The duration of the coarsening period increased with increasing temperature and the interface between the phases became sharp, characteristic of the late stages of phase separation. Because the ordering process continued after the target quench temperature was reached, the effective quench depth continued to increase after the initial phase separation. At high quench temperatures, the system was able to respond to the thermodynamic requirements of the increasing effective quench depth by undergoing secondary phase separation to form a droplet morphology within the preexisting bicontinuous one.     

IGF-I and IGFBP-3 transport in the rat heart

Specific binding of IGF-binding protein (IGFBP)-3 was shown to be present in the isolated, beating rat heart. The uptake of perfused (125)I-labeled IGF-I in the beating heart was decreased to 9% by blocking IGF-I binding sites with the IGF-I analog Long R(3) (LR(3)) IGF-I. When LR(3) was perfused with complexes of (125)I-IGF-I. IGFBP-3, uptake of (125)I-IGF-I was decreased to 41%, which was significantly greater than LR(3) and (125)I-IGF-I (41 vs. 9%). These data suggest that both microvessel IGF-I and IGFBP-3 binding sites contribute to the transport of IGF-I in the perfused rat heart. This also suggests a novel and plausible mechanism whereby circulating IGFs reach sites of IGF bioactivity.     

Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.     

Differential sensitivity to neurotensin-induced hypothermia, but not analgesia, in Fischer and Lewis rats

Neurotensin (NT) produces behavioral and physiological effects, including analgesia and hypotheria, when administered into the CNS. Fischer and Lewis rats exhibit differential behavioral responses to central NT receptor activation. To further characterize these differences, we assessed central NT-induced analgesia and hypothermia in independent groups of rats from each strain. Fischer and Lewis rats showed a similar dose-orderly analgesic response in a hot-plate test. Such an isosensitivity was not observed for NT-induced hypothermia. Although NT produced a dose-orderly decrease in mean rectal temperature in both strains, the magnitude of the hypothermic response was significantly smaller in Fischer than in Lewis rats. These findings provide further evidence of genetic differences in central neurotensinergeric neurotransmission in these two strains.     

Plasma 6beta-hydroxycortisol measurements for assessing altered hepatic drug metabolizing enzyme activity

To study the usefulness of 6beta-hydroxycortisol (6betaOHF) measurements for assessing hepatic drug metabolizing enzyme activity, plasma 6betaOHF and cortisol were measured in 22 patients with alcoholic liver disease after at least 2 weeks of alcohol abstinence, in 5 patients with severe Cushing's syndrome and in 12 healthy non-drinker subjects. Blood samples were drawn under resting conditions during midnight, in the morning at 0800 h, after a 1-mg overnight dexamethasone test and after ACTH administration. Plasma cortisol and 6betaOHF were determined with radioimmunoassay. In patients with alcoholic liver disease, the plasma cortisol levels at midnight and 0800 h, as well as after the administration of dexamethasone and ACTH were not different from corresponding values measured in non-drinker controls. In addition, these patients with alcoholic liver disease had similar plasma 6betaOHF levels at midnight, 0800 h and after dexamethasone administration as compared to corresponding values in controls. By contrast, ACTH administration in patients with alcoholic liver disease resulted in a significantly (p<0.05) larger increase of plasma 6betaOHF (from 106 +/- 22 to 1102 +/- 106 ng/dl, mean +/- SE) as compared to that found in controls (from 74 +/- 3 to 337 +/- 76 ng/dl). The markedly increased 6betaOHF response to ACTH administration in patients with alcoholic liver disease was similar to that measured in patients with severe Cushing's syndrome, in whom increased and non-suppressible plasma cortisol levels were accompanied by markedly elevated plasma 6betaOHF levels. These results indicate that alcohol abstinence in patients with alcoholic liver disease is associated with an exaggerated 6betaOHF response to ACTH and that this abnormality may prove to be a clinically useful parameter for a sensitive detection of altered drug metabolism present in these patients.