Publications

Human T-cell leukemia virus type I (HTLV-I) Rex and human immunodeficiency virus type 1 (HIV-1) Rev are essential gene products required for the replication of these two pathogenic human retroviruses. Both Rex and Rev act at a posttranscriptional level by binding to highly structured RNA-response elements, the Rex-response element in HTLV-I and the Rev-response element in HIV-1. Using a sensitive in vivo assay of protein-protein interaction, we now demonstrate that the HTLV-I Rex and HIV-1 Rev proteins readily form homomultimeric complexes in the absence of their cognate RNA-response elements yet fail to form heteromultimeric complexes with each other. Dominant negative mutations have been identified in both the rex and rev genes which presumably specify a critical activation or effector domain in each of these viral transactivators. Surprisingly, these dominant negative mutants of Rex and Rev fail to interact in vivo. These findings raise the possibility that the binding of nonfunctional monomers rather than functional multimers underlies the transdominant phenotype of these Rex and Rev mutants. Further, it seems likely that the assembly of functional and stable multimers of Rex and Rev in vivo may depend not only on the intrinsic multimerization domains of these proteins but also on the binding of a bridging cellular cofactor to the related activation domains present in each viral transactivator.

We conducted a review of the literature detailing the respiratory effects of chlorine, an extremely important but toxic halogen. Historically, the heaviest mass inhalational exposures to chlorine resulted from World War I gassing. Currently potential human exposure to chlorine inhalation occurs in a variety of settings in the workplace, as a result of inadvertent environmental releases, and even in the home due to household cleaning mishaps. Chlorine species are highly reactive; tissue injury results from exposure to chlorine, hydrochloric acid, hypochlorous acid, or chloramines. Acute, high level exposure to chlorine gas in occupational or environmental settings results in a variety of dose-related lung effects ranging from respiratory mucus membrane irritation to pulmonary edema. Pulmonary function testing can reveal either obstructive or restrictive deficits immediately following exposure, with resolution over time in the majority of cases. However, some of those exposed may demonstrate long-term persistent obstructive or restrictive pulmonary deficits or increased nonspecific airway reactivity after high level exposure to chlorine gas. Symptoms and signs following inhalation of mixtures of chlorine-containing cleaners in the home are similar to those after occupational exposures and environmental releases. Although generally less severe, these events may be extremely common. Controlled human exposure data suggest that some subjects may be more responsive to the effects of chlorine gas; epidemiologic data also indicate that certain subpopulations (e.g., smokers) may be at greater risk of adverse outcome after chlorine inhalation. Although these findings are intriguing, additional study is needed to better delineate the risk factors that predispose toward the development of long-term pulmonary sequelae following chlorine gas exposure.

Binding of IL-2 to the high affinity IL-2R results in the formation of a stable complex consisting of IL-2 and at least three distinct, noncovalently associated receptor subunits: IL-2R alpha (p55), IL-2R beta (p70-75), and IL-2R gamma (p64). Ligand binding also stimulates the rapid endocytosis of this receptor complex. To gain further insight into the function of the various subunits of the multicomponent high affinity IL-2R complex, we have carried out binding, internalization, and proliferation assays using an IL-2 analog designated F42A that contains a single Phe for Ala amino acid substitution at position 42. This mutation markedly reduces the intrinsic affinity of the resultant IL-2 analog for the low affinity IL-2R alpha subunit although having little or no effect on binding to the IL-2R beta (or IL-2R beta/gamma) intermediate affinity receptor. We have confirmed that F42A does not bind to the IL-2R alpha chain when expressed alone on MT-1 cells or in the presence of the large or small excess of IL-2R beta chains present on either YT-1 cells or forskolin-induced YT-1 cells, respectively. However, although F42A does not interact with the large number of low affinity IL-2R alpha chains present on HUT 102B2 cells, this ligand does bind to the small number of IL-2R beta chains present on these cells with at least 10-fold higher than expected affinity. These findings indicate that excess IL-2R alpha chains may exert positive effects on binding perhaps by changing the conformation of IL-2R beta. In F42A-stimulated internalization assays on forskolin-induced YT-1 cells, the IL-2R alpha chain is consistently endocytosed together with the IL-2R beta subunit indicating that IL-2R alpha is stably associated with the F42A-IL-2R beta complex even though the alpha-subunit contributes little if any affinity to the F42A binding reaction. In proliferation assays on mouse IL-3-dependent pro-B BA/F3 cells stably coexpressing transfected human IL-2R alpha and IL-2R beta subunits (but a mouse IL-2R gamma subunit), F42A proved to be a more effective agonist of growth than wild-type IL-2. These results suggest that the interaction between wild-type IL-2 but not F42A and the IL-2R alpha subunit in this mixed species high affinity receptor complex may induce an unfavorable receptor conformation leading to diminished rather than enhanced growth signal transduction.

Glomerular mesangial cells (MC) play a central role in the synthesis and turnover of the glomerular extracellular matrix. Prior studies [Davies, Thomas, Martin and Lovett (1988) Biochem. J. 251, 419-425; Martin, Davies, Thomas and Lovett (1989) Kidney Int. 36, 790-801] have characterized at the protein level a 72 kDa type IV collagenase that is secreted by cultured human and rat MC. While exposure of most cell types to interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) or phorbol ester has little or even an inhibitory effect on 72 kDa type IV collagenase secretion, these factors significantly increased the synthesis of this enzyme by rat MC. Given this divergent pattern of expression, a homology-based PCR cloning strategy using rat MC cDNA templates was employed to define at the molecular level the structure of the mesangial 72 kDa type IV collagenase. The nucleotide sequence within the open reading frame of the rat mesangial 72 kDa type IV collagenase cDNA diverges from the sequence of the human homologue by approx. 9%. The divergence in the 3' untranslated region was much more extensive. Steady-state levels of the 3.1 kb transcript of the 72 kDa type IV collagenase were low or undetectable in resting MC, but were greatly stimulated following incubation with IL-beta, TNF-alpha or phorbol ester. None of these factors induced synthesis by MC of the closely related 92 kDa type IV collagenase. Synthesis by MC of the 72 kDa type IV collagenase was also induced by second-messenger analogues, including 8-bromo-cyclic AMP and forskolin. It is concluded that MC regulate the expression of this enzyme in an unusual, tissue-specific fashion. Cytokine and second-messenger inducibility may contribute to the enhanced expression of the enzyme during glomerular inflammatory disorders.