SummaryOpportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-~B in response to a variety of stimuli in T and B cells. The effect ofgliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56 lck and p59 fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-~B DNA binding in vitro. However, in intact cells, inhibition of NF-~13 did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IKB-ci, NF-KB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-KB inhibition.

Hypoxia is a pathophysiological condition that occurs during injury, ischemia, and stroke. It is characterized by a decrease of reactive oxygen intermediates and a change of the intracellular redox level. In tumors hypoxia is regarded as a trigger for enhanced growth and metastasis. Here we report that in HeLa cells, hypoxic conditions induce the transcriptional activation of c-fos transcription via the serum response element. Mutations in the binding site for the ternary complex factor Elk-1 and the serum response factor abolished this induction, indicating that a ternary complex at the serum response element is necessary for the induction of the c-fos gene under hypoxia. The transcription factor Elk-1 was covalently modified by phosphorylation in response to hypoxia. Furthermore this hyperphosphorylation of Elk-1, the activation of mitogen-activated protein kinase (MAPK), and the induction of c-fos transcripts were blocked by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase 1. An in vitro kinase assay with Elk-1 as substrate showed that MAPK is activated under hypoxia. The activation of MAPK corresponds temporally with the phosphorylation and activation of Elk-1. Thus, a decrease of the intracellular reactive oxygen intermediate level by hypoxia induces c-fos via the MAPK pathway. These results suggest that the intracellular redox levels may be directly coupled to tumor growth, invasion, and metastasis via Elk-1-dependent induction of c-Fos controlled genes.The activation of c-fos by mitogens and changes of the intracellular redox level is mainly mediated by the serum response element (SRE).1 The SRE is a regulatory element found in many growth factor-regulated promotors that directs the rapid induction of gene expression (for review see Ref. 1). The best studied SRE is that of the c-fos gene. Two kinds of transcription factors are required for SRE activity: the ubiquitous transcription serum response factor (SRF) (2) and the ternary complex factors (TCFs), which form a ternary complex with the SRF. The human TCFs include Elk-1, SAP-1, and SAP-2 and constitute a subfamily within the Ets family of transcription factors (for review see Ref.3). These proteins need SRF to bind tightly to the SRE (4). The N-terminal domains of Elk-1 and SAP-1 mediate DNA contact and ternary complex formation. The transactivation domain at the C terminus contains several conserved MAPK phosphorylation sites. Growth factor-stimulated activation of the MAPK pathway results in phosphorylation of the Elk-1 C terminus (5), which then cooperates with the SRF C-terminal activation domain to activate transcription (6, 7).The transcription factor AP-1 is composed of members of the Fos family (c-Fos, Fos-B, Fra-1, and Fra-2) and the Jun family (c-Jun, JunB, and JunD) that form restricted homo-or heterodimers (for review see Ref. 8). With the exception of c-Jun homodimers, AP-1 is predominantly induced at a transcriptional level by novel synthesis of its subunits. Thi...

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

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