The 3' untranslated region of IL-1beta regulates protein production.

IL-1beta, a pro-inflammatory cytokine, has sequences in its 3' untranslated region (UTR) that may play a role in the post-transcriptional regulation of IL-1beta production. To test this hypothesis, a series of chimeric reporter genes were developed consisting of a chloramphenicol acetyltransferase (CAT) gene the native 3'-UTR of which was replaced by the full-length IL-1beta 3'-UTR or various 3'-UTR deletion mutants. Expression of these constructs under the SV40 late promoter in THP-1 cells showed that the full-length 3'-UTR repressed constitutive CAT activity to 28% of control CAT activity. Further analysis of the 3'-UTR localized the repressor signal to an adenosine-thymidine (AdT)-rich region. Upon exposure to LPS, the full-length IL-1beta 3'-UTR mediated almost a fivefold increase in CAT activity. The LPS response was not simply loss of AdT-mediated repression; when this sequence was tested alone, it did not respond to LPS. The LPS response effect was localized to the terminal 177 base pairs of the IL-1beta 3'-UTR. The increase in CAT activity following LPS stimulation was associated with an increased CAT.IL-1beta 3'-UTR mRNA half-life, indicating at least one effect of the 3'-UTR on a post-transcriptional process. These studies demonstrate that the IL-1beta 3'-UTR is involved in the regulation of IL-1beta protein production, and a LPS response element may be in the IL-1beta 3'-UTR.

A partition coefficient determination method for nonvolatile chemicals in biological tissues.

Partition or distribution coefficients are critical elements in efforts designed to describe the uptake, distribution, biotransformation, and excretion of organic chemicals in biological systems. In order to estimate the partition coefficients needed to describe the biological distribution of low-volatility compounds, an experimental method was developed to measure partitioning of nonvolatile compounds into biological tissues. Blood, fat, muscle, liver, and skin were individually incubated in a saline solution containing the chemical of interest. Each sample was centrifuged and 2.0 ml of the supernatant was removed and placed into a prewashed, low binding 10,000 MW cutoff Millipore filter cell. Each cell was fitted with a magnetic stirrer and 32 psi nitrogen was applied to the closed cell. The filtrate was collected, extracted, and analyzed for the chemical of interest. The chemicals evaluated were parathion, lindane (hexachlorocyclohexane), paraoxon, perchloroethylene, trichloroacetic acid, and dichloroacetic acid. These chemicals were chosen to develop this method because their vapor pressures range from 9 x 10(6) to 14.2 mm Hg at 20 degrees C. For the one volatile chemical evaluated, perchloroethylene, the method provided partition coefficient results that were in good agreement with values obtained using the vial equilibration method. The nonvolatile partition coefficient method described in this paper demonstrates an approach for evaluation of chemicals with diverse chemical structure and solubility properties.

Heat stress regulates the human 70-kDa heat-shock gene through the 3'-untranslated region.

Cells respond to a variety of stresses by synthesizing a family of proteins termed heat-shock proteins (HSP). Recently, the 3'-untranslated regions (UTRs) of some mRNAs have been shown to be important in the posttranscriptional regulation of protein production. Therefore, we hypothesized that heat could regulate HSP70 production through the HSP70 3'-UTR, in addition to its known effects on transcription. To test this hypothesis, cells were transfected with either a plasmid containing sequences encoding the human HSP70 or beta-globin 3'-untranslated region placed downstream of a chloramphenicol acetyltransferase (CAT) reporter gene. In both plasmids, the CAT gene was driven by an SV40 promoter. Following heat stress, cells transfected with the CAT construct containing the HSP70 3'-UTR showed increased CAT activity relative to the beta-globin 3'-UTR construct. This effect paralleled increases in HSP70 mRNA and levels of the inducible HSP70 protein by Western blot. These studies identify a heat-induced mechanism of posttranscriptional control of HSP70 synthesis utilizing the HSP70 3'-UTR, which may be important in the cells ability to regulate the heat-shock response.