Thyroid hormone kinetics during late pregnancy in the ovine fetus

The factors responsible for the changes in the plasma concentrations of thyroid hormones in the ovine fetus in late pregnancy were investigated by making serial measurements of the concentrations, metabolic clearance rates and production rates of T3 and T4 in 17 fetuses. The concentrations of T3 in fetuses of 135-145 days gestational age were four times higher than in those of 110-125 days but the concentrations of T4 were unchanged. The metabolic clearance rate of T3 halved over this period whereas that of T4 rose slightly. The production rate of T3 more than doubled and of T4 increased slightly but not significantly. We conclude that the concentration of T4 shows little change with increasing gestational age because the trends in metabolic clearance rates and production rates are weak and in the same direction. The sharp rise in the concentration of T3 is attributable to a fall in metabolic clearance rate coupled with a rise in production rate.     

Hepatocellular kinetics and the expression of growth hormone (GH) in the livers and liver tumours of GH-transgenic mice

Although it is well known that transgenic mice that overexpress growth hormone (GH) frequently develop liver tumours, the precise nature of the relationship between the overexpression of GH and hepatocarcinogenesis is not clear. The current study was designed to investigate the relationship between the expression of the GH transgene and changes in hepatocyte morphology and kinetics, prior to and during hepatocarcinogenesis in GH-transgenic mice. In young mice (1-month-old) prior to tumour development, GH protein, as detected by immunohistochemistry, was observed in the cytoplasm of essentially all hepatocytes. In liver tissues of older animals, apoptotic cells and hypertrophic hepatocytes did not express immunoreactive GH, even though GH was expressed strongly in the smaller hepatocytes. A relatively high proportion of large dysplastic hepatocytes (>50 microm) were apoptotic (TUNEL positive), whereas smaller hepatocytes featured more prominently in the proliferative phase, as measured by BrdU incorporation. GH expression in tumour tissue, as detected by immunohistochemistry, was often variable and generally decreased with tumour development. Northern blot analysis showed that equivalent levels of GH mRNA were present in tumour tissue and adjacent liver. However, there was no clear trend when the levels of GH mRNA extracted from adenoma, and hepatocellular carcinoma, were compared. These observations help clarify some of the mechanisms by which GH promotes hepatocarcinogenesis in GH-transgenic mice.     

Molecular size of the nuclear thyroid hormone receptor

Among the previously reported putative nuclear thyroid hormone receptor forms having molecular masses of 56-59 kDa and 45-49 kDa, respectively, only the former can be the endogenous receptor. The latter must be a degradation product because it is virtually absent in rat liver nuclear extracts prepared in the presence of 20% glycerol and 5 mM Mg2+, which inhibit degradation. In the absence of glycerol, the receptor form of lower mass was present in large amounts in nuclear extracts. Sucrose could not replace glycerol as a protective agent, even in the presence of Mg2+. Thus, the endogenous nuclear thyroid hormone receptor appears to be labile under the experimental conditions used in preparing nuclear extracts. The molecular mass of the nuclear receptor was determined to be 57 kDa on the basis of SDS-polyacrylamide gel electrophoresis after photoaffinity labeling of nuclear proteins with (3,5-125I)-labeled thyroxine.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.     

Maternal hormone levels during Na 872 infusion in late pregnancy

In order to assess the effects of Na 872 infusion on maternal endocrinology during late pregnancy, we measured serum estradiol, estriol, progesterone, human placental lactogen, follicle simulating hormone, prolactin, cyclic adenosine monophosphate, cortisol and insulin before, during and after Na 872 infusion (bromhexine 2 mg/min for one hour) in ten mothers. As compared to six control mothers, no changes could be observed. This suggests that the claimed maturing effect of Na 872 on the fetal lungs is not mediated by the hormonal changes measurable in maternal blood.     

Differential gene expression of livers from ApoE deficient mice

A genomic survey for differentially expressed genes was performed to livers of ApoE deficient mice using human cDNA microarray containing approximately 9,000 human cDNA clones. Due to the homology between mouse and human, hybridization was performed at lower stringency condition, 10 degrees below the regular hybridizing temperature. Gene expression profiles of livers corresponding to high levels of blood cholesterol were generated at genomic scale. Thirty-seven genes were randomly selected from a pool of differentially expressed genes and subjected to semi-quantitative RT-PCR, further confirmed the result from microarray hybridization. These included genes associated with atherosclerosis, and novel genes that implied novel pathways correlated to high levels of blood cholesterol. It is promising using human cDNA microarray, the most complete collection among all species, to study other mammalian systems with satisfying speed and accuracy.     

Up-regulation of orphan nuclear estrogen-related receptor alpha expression during long-term caloric restriction in mice

The estrogen-related receptor alpha (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily that regulates a number of target genes encoding enzymes that participate in various metabolic pathways involved in maintaining energy balance in animals. In this study, whether long-term caloric restriction (alternate days of fasting for 3 months) in mice modulates the expression of ERRα in various tissues was investigated. Western blot analyses showed positive immunoreactive ERRα protein (53 kDa) band in various mice tissue extracts, though at varying levels. Heart, kidney, and skeletal muscles expressed significant levels of ERRα, with a comparatively lower level detected in the intestine, brain, and liver. Cardiac ERRα expression was the highest, with the least detected in the liver. Caloric restricted mice exhibited a significant increase in ERRα level in the heart (5.45-fold), kidney (3.70-fold), skeletal muscle (3.0-fold), small intestine (2.72-fold), and liver (2.44-fold) extracts as compared to ad libitum fed. However, caloric restriction could not evoke any detectable receptor level change in the brain. Notably, the highest ERRα up-regulation was detected in the heart. This up-regulation in ERRα level especially in highly oxidative tissues such as heart, kidney, small intestine, and skeletal muscle of caloric restricted mice may be helpful in modulating ERRα responsive genes that participates in maintaining energy balance. This may potentially strengthen the metabolic and biochemical adaptation in such tissues, which is necessary for animal survival under long-term caloric restriction.     

Decreased expression of protease-activated receptor 4 in human gastric cancer

Protease-activated receptors (PARs) are a unique family of G-protein coupled receptors. PAR4, the most recently identified PAR member, was reported to be overexpressed during the progression of colon and prostate cancers. Though PAR4 mRNA was detected in normal stomach, the role of PAR4 in gastric cancer has not been investigated. In this study, differential expression of PAR4 was measured by real-time PCR (n=28) and tissue microarrays (n=74). We showed that PAR4 was located from basal to middle portions of normal gastric mucosa. PAR4 expression was remarkably decreased in gastric cancer tissues as compared with matched noncancerous tissues, especially in positive lymph node or low differentiation cancers. Furthermore, methylation of the PAR4 promoter in cell lines was assessed by treatment with 5-aza-2'-deoxycytidine and genomic bisulfite sequencing. AGS and N87 human gastric cancer cell lines did not express PAR4, as compared to HT-29 human colon cancer cell line with significant PAR4 expression. Treatment with 5-aza-2'-deoxycytidine restored PAR4 expression in AGS and N87 cells, which exhibited significantly more 5-methylcytosines in the PAR4 promoter compared with HT-29 cells. Our results revealed that down-regulation of PAR4 expression occurs frequently in gastric cancers and exhibits association with more aggressive gastric cancer. Interestingly, the loss of PAR4 expression in gastric cancers may result from hypermethylation of the PAR4 promoter.     

Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.