Tuning Plant Promoters Using a Simple Split Luciferase Method to Assess Transcription Factor-DNA Interactions.

Sequence features, including the affinity of binding motifs for their cognate transcription factors, are important contributors to promoter behavior. The ability to predictably recode affinity enables the development of synthetic promoters with varying levels of response to known cellular signals. Here we describe a luminescence-based microplate assay for comparing the interactions of transcription factors with short DNA probes. We then demonstrate how these data can be used to design synthetic plant promoters of varying strengths that respond to the same transcription factor.

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands.

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1-4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono-fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.

Transdermal delivery of narcotic analgesics: comparative metabolism and permeability of human cadaver skin and hairless mouse skin.

The permeation of hairless mouse skin and human cadaver skin by narcotic analgesics was investigated to determine the interspecies variation. Permeability coefficients of morphine, fentanyl, and sufentanil across full-thickness hairless mouse skin were 1 order of magnitude higher than those found for human epidermis. The permeability coefficient of morphine for stripped hairless mouse skin was 500-fold higher than that for intact skin, showing the stratum corneum to be the principal barrier to its penetration. The permeability coefficient of fentanyl for stripped hairless mouse skin was also raised, but stripping caused an inappreciable increase in the permeation rate of sufentanil. The thick dermis of excised mouse skin obviously offered a significant resistance to the permeation of these lipophilic compounds. In comparison, the permeability coefficients of fentanyl and sufentanil through stripped cadaver epidermis (n > or = 25) were 67 and 37 higher than for intact human epidermis, respectively. The skin metabolism of the narcotics was investigated. No significant metabolic degradation of morphine, fentnayl, and sufentanil was observed in either fresh human cadaver skin or hairless mouse skin homogenates in the presence of NADPH cofactor, suggesting a low monooxygenase enzyme presence in skin. Moreover, no measurable glucuronidation of morphine took place in human skin or hairless mouse skin. Both processes proceeded rapidly in liver homogenates (mouse) under identical circumstances. It thus appears that these drugs pass through in intact form.

Investigation of guanine-nucleotide-binding protein involvement and regulation of cyclic AMP metabolism in interleukin 1 signal transduction.

The involvement of guanine-nucleotide-binding proteins (G-proteins) and regulation of cyclic AMP (cAMP) in interleukin 1 (IL1) signal transduction has been investigated in EL4 and 7OZ/3 cells expressing Type 1 and Type 2 IL1 receptors respectively. Results show that in both cell types IL1 alone failed to induce changes in cellular cAMP levels, and in membrane preparations the cytokine had no significant effect on adenylate cyclase activity. In contrast, forskolin stimulated cAMP levels in cells and membranes. IL1 did not significantly alter GTPase activity or rate of guanosine 5'-[gamma-[35S]thio]triphosphate binding measured in membrane preparations from the EL4 and 7OZ/3 cells. In EL4-cell membrane preparations the kinetics of 125I-IL1 binding were altered in the presence of guanosine 5'-[beta gamma-imido]triphosphate, resulting in the formation of a higher-affinity state for IL1 binding. Adenosine 5'-[beta gamma-imido]triphosphate at the same concentration was without effect. These results suggest that IL1 receptor function may be regulated by guanine nucleotides; however, the mechanism appears to differ from that exhibited by conventional G-protein-linked receptors. The lack of significant effects of IL1 on cAMP metabolism in these cells suggests that alternative pathways must exist to mediate the intracellular responses to stimulation via both types of the IL1 receptor.

Analysis of mutations in the putative nuclear localization sequence of interleukin-1 beta.

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210----Ala), M50 (Lys211----Ala) and M51 (Pro208----Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.

Modification of biological responses to interleukin-1 by agents that perturb signal transduction pathways.

In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-1). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular cAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-1-induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E2. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-1, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate.