We describe a noninvasive, quantitative, and tomographic method to visualize lymphocytes within the whole animal. We used positron-emission tomography (PET) to follow the localization of adoptively transferred immune T lymphocytes. Splenic T cells from animals that had rejected a Moloney murine sarcoma virus͞ Moloney murine leukemia virus (M-MSV͞M-MuLV)-induced tumor were marked with a PET reporter gene, injected into tumor-bearing mice, and imaged in a microPET by using a substrate specific for the reporter. Specific localization of immune T cells to the antigenpositive tumor was detected over time, by sequential imaging of the same animals. Naive T cells did not localize to the tumor site, indicating that preimmunization was required. Autoradiography and immunohistochemistry analysis corroborated the microPET data. The method we have developed can be used to assess the effects of immunomodulatory agents intended to potentiate the immune response to cancer, and can also be useful for the study of other cell-mediated immune responses, including autoimmunity.

In the purple, photosynthetic bacterium, Rhodobacter capsulatus, the RegB/RegA two-component system is required for activation of several anaerobic processes, such as synthesis of the photosynthetic apparatus and assimilation of CO 2 and N 2 . It is believed that RegB is an integral membrane histidine kinase that monitors the external environment. Under anaerobic growth conditions, it transduces a signal through phosphorylation of the response regulator, RegA, which then induces target gene expression. We used an in vitro assay to characterize the phosphorylation of wild-type RegA and a mutant variant (RegA*) that is responsible for abnormally high photosynthesis gene expression under both aerobic and anaerobic growth conditions. Phosphorylation assays indicate that phosphorylated RegA* (RegA*ϳP) is much more stable than RegAϳP, indicating that it may be locked in a conformation that is resistant to dephosphorylation. DNase I footprint assays also indicate that unphosphorylated RegA* has a much higher affinity for specific DNA binding sites than the wild-type protein.Phosphorylation of RegA* increases DNA binding 2.5-fold, whereas phosphorylation of RegA increases DNA binding more than 16-fold. Collectively, these results support the hypothesis that RegA* is a constitutively active variant that does not require phosphorylation to assume a structural conformation required to bind DNA.

AKR1C2, also referred to as the human bile acid binder and 3␣-hydroxysteroid dehydrogenase type III, is a multifunctional oxidoreductase able to stereoselectively reduce steroids as well as oxidize or reduce polyaromatic hydrocarbons. Previously, this same protein was also identified by its robust induction by phase II inducers in HT29 cells. In HepG2 cells, both AKR1C2 and AKR1C1 (97% sequence homology) were induced by phase II inducers but not the highly related AKR1C3 and AKR1C4 family members (84% sequence homology). We now report the initial characterization of the proximal promoter of AKR1C2 in HepG2 cell line and the identification of a potent enhancer-like element responsive to phase II inducers located approximately 5.5 kilobases upstream from the transcription start site. DNA sequence analysis of this enhancer element revealed that it contained a consensus antioxidant response element (ARE), which was confirmed by mutation analysis.Treatment with phase II inducers leads to increased accumulation of nuclear factor-erythroid 2 p45-related factor (NRF) 2 in the nucleus, which was associated with increased binding to this ARE as determined by electrophoretic mobility shift assay. Transient transfection with Nrf2 increased the transcriptional activity of the ARE of AKR1C2 comparable with that observed with phase II inducers. Chromatin immunoprecipitation (ChIP) analysis also confirmed increased NRF2 binding to the ARE after induction by a phase II inducer. The AKR1C1 promoter also harbored this same ARE element in a highly homologous region, which was also bound by NRF2 in a ChiP analysis. No induction of the ARE of AKR1C2 was detected in Nrf2fibroblasts. The regulation of AKR1C2 by this distal ARE suggests that AKR1C2 detoxifies products of reactive oxidant injury, which has important implications for both hormone and xenobiotic metabolism.

SummaryIt has been known for over half a century that anoxygenic photosynthetic bacteria maximally synthesize their photosystems in the absence of oxygen. During the last decade, it has become clear that this regulation is largely at the transcriptional level, with photosynthesis genes expressed only under anaerobic conditions. We describe here in vitro reconstitution of activation and repression of three photosynthesis promoters, bch (bacteriochlorophyll biosynthesis), puc (light-harvesting II apoproteins) and puf (reaction centre and light-harvesting I apoproteins) using puri®ed transcription factors and RNA polymerase from Rhodobacter capsulatus. Previous genetic results have indicated that each of these three promoters is differentially regulated by three key regulators: CrtJ acting as a repressor of bch and puc and the two-component regulators RegA/RegB, which are activators of puc and puf. These regulators are distinct from those that mediate oxygen control in enteric bacteria. Our in vitro studies show that these puri®ed regulators directly control the expression of the housekeeping RNA polymerase at these promoters. High-level basal expression of the bch promoter is shown to be repressed by CrtJ. The puc promoter is activated by the RegB-phosphorylated RegA protein and additionally repressed by CrtJ. At the puc promoter, CrtJ effectively competes for promoter binding with RegA, while at the bch promoter, repression appears to be by competition for the RNA polymerase binding site. In contrast to what has been suggested previously, the RegA-activated puf promoter is demonstrated as being recognized by the housekeeping RNA polymerase. We also discuss evidence that RegA,P activation of the puc and puf promoters involves recruitment of RNA polymerase by different modes of protein±protein interaction.