Cytochrome b(5) coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium.

Addition of cytochrome b(5) to recombinant cytochrome P450 2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro. To determine if this effect could be observed with recombinant expression systems in vivo, we have constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b(5) in Salmonella typhimurium lacking ogt and ada methyltransferases (YG7104, ogt(-); and YG7108, ogt(-), ada(-)). These new recombinant strains exhibit a four- to five-fold greater mutagenic response to dimethylnitrosamine, diethylnitrosamine, and dipropylnitrosamine than strains that contain only CYP2E1 and reductase, and are over 100-fold more sensitive to nitrosamines than the parental strains in the presence of an exogenous activating system (S9 fraction). The four-fold increase in mutagenicity in the presence of cytochrome b(5) was consistent with increasing alkyl chain length up to dibutylnitrosamine, which was poorly activated by CYP2E1. The greatest enhancement was obtained with a tricistronic construct in which the b(5) cDNA preceded the P450 and reductase cDNAs; placing the b(5) cDNA after the reductase cDNA was substantially less effective. These new, highly sensitive strains may prove useful in the detection of nitrosamine contamination of food and environmental samples.

R7 photoreceptor specification requires Notch activity.

The eight photoreceptors in each ommatidium of the Drosophila eye are assembled by a process of recruitment [1,2]. First, the R8 cell is singled out, and then subsequent photoreceptors are added in pairs (R2 and R5, R3 and R4, R1 and R6) until the final R7 cell acquires a neuronal fate. R7 development requires the Sevenless receptor tyrosine kinase which is activated by a ligand from R8 [3]. Here, we report that the specification of R7 requires a second signal that activates Notch. We found that a Notch target gene is expressed in R7 shortly after recruitment. When Notch activity was reduced, the cell was misrouted to an R1/R6 fate. Conversely, when activated Notch was present in the R1/R6 cells, it caused them to adopt R7 fates or, occasionally, cone cell fates. In this context, Notch activity appears to act co-operatively, rather than antagonistically, with the receptor tyrosine kinase/Ras pathway in R7 photoreceptor specification. We propose two models: a ratchet model in which Notch would allow cells to remain competent to respond to sequential rounds of Ras signalling, and a combinatorial model in which Notch and Ras signalling would act together to regulate genes that determine cell fate.

Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase.

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds is problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. We have constructed two mutagenicity tester strains that co-express full-length human cytochrome P450 2E1 and P450 reductase in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt- and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar to or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. These strains were also sensitive to nitrosamines with longer alkyl side chains including diethylnitrosamine, dipropylnitrosamine and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.

Spatially restricted factors cooperate with notch in the regulation of Enhancer of split genes.

Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.

Photofrin photodynamic therapy can significantly deplete or preserve oxygenation in human basal cell carcinomas during treatment, depending on fluence rate.

At high fluence rates in animal models, photodynamic therapy (PDT) can photochemically deplete ambient tumor oxygen through the generation of singlet oxygen, causing acute hypoxia and limiting treatment effectiveness. We report that standard clinical treatment conditions (1 mg/kg Photofrin, light at 630 nm and 150 mW/cm2), which are highly effective for treating human basal cell carcinomas, significantly diminished tumor oxygen levels during initial light delivery in a majority of carcinomas. Oxygen depletion could be found during at least 40% of the total light dose, but tumors appeared well oxygenated toward the end of treatment. In contrast, initial light delivery at a lower fluence rate of 30 mW/cm2 increased tumor oxygenation in a majority of carcinomas. Laser treatment caused an intensity- and treatment time-dependent increase in tumor temperature. The data suggest that high fluence rate treatment, although effective, may be inefficient.

Suppressive immunization with DNA encoding a self-peptide prevents autoimmune disease: modulation of T cell costimulation.

Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive from of vaccination, with DNA encoding a minigene for residues 139-151 of myelin proteolipid protein (PLP139-151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-gamma, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-gamma were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139-151 reduced the capacity of a T cell line reactive to PLP139-151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.

Frizzled regulation of Notch signalling polarizes cell fate in the Drosophila eye.

The Drosophila eye, a paradigm for epithelial organization, is highly polarized with mirror-image symmetry about the equator. The R3 and R4 photoreceptors in each ommatidium are vital in this polarity; they adopt asymmetrical positions in adult ommatidia and are the site of action for several essential genes. Two such genes are frizzled (fz) and dishevelled (dsh), the products of which are components of a signalling pathway required in R3, and which are thought to be activated by a diffusible signal. Here we show that the transmembrane receptor Notch is required downstream of dsh in R3/R4 for them to adopt distinct fates. By using an enhancer for the Notch target gene Enhancer of split mdelta, we show that Notch becomes activated specifically in R4. We propose that Fz/Dsh promotes activity of the Notch ligand Delta and inhibits Notch receptor activity in R3, creating a difference in Notch signalling capacity between R3 and R4. Subsequent feedback in the Notch pathway ensures that this difference becomes amplified. This interplay between Fz/Dsh and Notch indicates that polarity is established through local comparisons between two cells and explains how a signal from one position (for example, the equator in the eye) could be interpreted by all ommatidia in the field.

Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis.

RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis.

Cutaneous phototoxic occurrences in patients receiving Photofrin.

Incidence of cutaneous phototoxic reactions induced by intravenous injection of Photofrin polyporphyrin was assessed in a series of 180 patients (266 injections) undergoing photodynamic therapy (PDT) at Roswell Park Cancer Institute during the period 1986-1989. In addition to the usual verbal questions regarding phototoxic reactions solicited at follow-up, forty-two patients in this group also responded to a written questionnaire designed to solicit answers to specific questions. Photofrin doses ranged from 0.5 to 2.0 mg/kg. Overall, 20-40% of patients reported some type of phototoxic response.