Binding of radioactive thyroxine to proteins in the plasma of vertebrates was studied by electrophoresis followed by autoradiography. Albumin was found to be a thyroxine carrier in the blood of all studied fish, amphibians, reptiles, monotremes, marsupials, eutherians (placental mammals), and birds. Thyroxine binding to transthyretin was detected in the blood of eutherians, diprotodont marsupials, and birds, but not in blood from fish, toads, reptiles, monotremes, and Australian polyprotodont marsupials. Globulins binding thyroxine were only observed in the plasma of some mammals. Apparently, albumin is the phylogenetically oldest thyroxine carrier in vertebrate blood. Transthyretin gene expression in the liver developed in parallel, and independently, in the evolutionary lineages leading to eutherians, to diprotodont marsupials, and to birds. In contrast, high transthyretin mRNA levels, strong synthesis, and secretion of transthyretin in choroid plexus from reptiles and birds indicate that transthyretin gene expression in the choroid plexus evolved much earlier than in the liver, probably at the stage of the stem reptiles. NH2-terminal sequence analysis suggests a change of transthyretin pre-mRNA splicing during evolution.

Glucocorticoid (GC) hormones cause pronounced T cell apoptosis, particularly in immature thymic T cells. This is possibly due to tissue-specific regulation of the glucocorticoid receptor (GR) gene. In mice the GR gene is transcribed from five separate promoters designated: 1A, 1B, 1C, 1D, and 1E. Nearly all cells express GR from promoters 1B–1E, but the activity of the 1A promoter has only been reported in the whole thymus or lymphocyte cell lines. To directly assess the role of GR promoter use in sensitivity to glucocorticoid-induced cell death, we have compared the activity of the GR 1A promoter with GC sensitivity in different mouse lymphocyte populations. We report that GR 1A promoter activity is restricted to thymocyte and peripheral lymphocyte populations and the cortex of the brain. The relative level of expression of the 1A promoter to the 1B–1E promoters within a lymphocyte population was found to directly correlate with susceptibility to GC-induced cell death, with the extremely GC-sensitive CD4+CD8+ thymocytes having the highest levels of GR 1A promoter activity, and the relatively GC-resistant αβTCR+CD24int/low thymocytes and peripheral T cells having the lowest levels. DNA sequencing of the mouse GR 1A promoter revealed a putative glucocorticoid-response element. Furthermore, GR 1A promoter use and GR protein levels were increased by GC treatment in thymocytes, but not in splenocytes. These data suggest that tissue-specific differences in GR promoter use determine T cell sensitivity to glucocorticoid-induced cell death.

Xenopus laevis transthyretin (xTTR) cDNA was cloned and sequenced. The derived amino acid sequence was very similar to those of other vertebrate transthyretins (TTR). TTR gene expression was observed during metamorphosis in X. laevis tadpole liver but not in tadpole brain nor adult liver. Recombinant xTTR was synthesized in Pichia pastoris and identified by amino acid sequence, subunit molecular mass, tetramer formation, and binding to retinol-binding protein. Contrary to mammalian xTTRs, the affinity of xTTR was higher for L-triiodothyronine than for L-thyroxine. The regions of the TTR genes coding for the NH(2)-terminal sections of the polypeptide chains of TTR seem to have evolved by stepwise shifts of mRNA splicing sites between exons 1 and 2, resulting in shorter and more hydrophilic NH(2) termini. This may be one molecular mechanism of positive Darwinian evolution. Open reading frames with xTTR-like sequences in the genomes of C. elegans and several microorganisms suggested evolution of the TTR gene from ancestor TTR gene-like "DNA modules." Increasing preference for binding of L-thyroxine over L-triiodothyronine may be associated with evolving tissue-specific regulation of thyroid hormone action by deiodination.

Background: A rapid and comprehensive metabolic stability screen at the top of a drug discovery flow chart serves as an effective gate in eliminating low value compounds. This imparts a significant level of efficiency and saves valuable resources. While microsomes are amenable to high throughput automation and are cost effective, their enzymatic make-up is limited to that which is contained in endoplasmic reticulum, thereby informing only on Phase I metabolism. Lack of Phase II metabolism data can become a potential liability later in the process, adversely affecting discovery projects’ timelines and budget. Hepatocytes offer a full complement of metabolic enzymes and retain their cellular compartments, better representing liver metabolic function. However, hepatocyte screens are relatively expensive, labor intensive, and not easily automatable. Liver S9 fractions include Phase I and II metabolic enzymes, are relatively inexpensive, easy to use, and amenable to automation, making them a more appropriate screening system. We compare the data from the three systems and present the results.Results: Liver S9 and hepatocyte stability assays binned into the same category 70-84% of the time. Microsome and hepatocyte data were in agreement 73-82% of the time. The true rate for stability versus plasma clearance was 45% for hepatocytes and 43% for S9.Conclusion: In our opinion, replacing liver microsome and hepatocyte assays with S9 assay for high throughput metabolic screening purposes provides the combined benefit of comprehensive and high quality data at a reasonable expense for drug discovery programs.

Structure and function were studied for Crocodylus porosus transthyretin (crocTTR), an important intermediate in TTR evolution. The cDNA for crocTTR mRNA was cloned and sequenced and the amino acid sequence of crocTTR was deduced. In contrast to mammalian TTRs, but similar to avian and lizard TTRs, the subunit of crocTTR had a long and hydrophobic NH(2)-terminal region. Different from the situation in mammals, triiodothyronine (T(3)) was bound by crocTTR with higher affinity than thyroxine (T(4)). Recombinant crocTTR and a chimeric construct, with the NH(2)-terminal region of crocTTR being replaced by that of Xenopus laevis TTR, were synthesized in the yeast Pichia pastoris. Analysis of the affinity of the chimeric TTRs showed that the NH(2)-terminal region modulates T(4) and T(3) binding characteristics of TTR. The structural differences of the NH(2)-terminal regions of reptilian and amphibian TTRs were caused by a shift in splice sites at the 5' end of exon 2. The comparison of crocodile and other vertebrate TTRs shows that TTR evolution is an example for positive Darwinian evolution and identifies its molecular mechanism.