Residues near the amino terminus of Rns are essential for positive autoregulation and DNA binding

Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.     

The Relationship between myo-Inositol, ATP and Rotational Streaming

The rotational streaming of cytoplasm in barley root hairs has been stimulated about 1.3–1.8 times through continuous treatment with various solutions of myo-inositol. The stimulation attained the same level as after ATP administration and was dependent on the external myo-inositol supply with the employed concentrations. The stimulation was cut off by simultaneous treatment with myo-inositol and uranyl salts. By using uranyl acetate the rate of streaming was maintained about the value of the control. The uranyl chloride caused an inhibition in the rotational streaming and later made it to cease altogether. The simultaneous treatment with myo-inositol and 2.4-DNP (dinitrophenol) induced a quick inhibition in 60% of the root hairs. consecutively stopping rotational streaming. It is assumed that the stimulation of rotational streaming is not due to the direct effect of myo-inositol but to ATP formed in the reaction: inositol hexaphosphate ++ ADP ? ATP + inositol. According to the results obtained by testing with uranyl salts, the phosphorylation of inositol probably takes place at the cell surface. The effect of 2,4-nNP points to the presence of two competitive metabolic processes involved in ATP consumption: the upkeep of rotational streaming and the uptake of substances by the plant cell.     

Does hypothyroidism augment sun-induced skin damage?

Objectives: We investigated the mutual effects of overt hypothyroidism and prolonged sunlight exposure on free radical accumulation and oxidative skin damage.

Methods: Free radical accumulation was evaluated by monitoring the transformation of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) into MTT-formazan. The pro-oxidant enzymes xanthine oxidase (XO) and NADPH-diaphorase were measured in the skin. XO activity was estimated based on the yield of uric acid, while NADPH-diaphorase reactivity was monitored histochemically as an indirect marker of nitric oxide synthase and nitric oxide activity. Cellular damage was determined by malondialdehyde formation, a marker for lipid peroxidation.

Results: In the skin of both euthyroid and hypothyroid animals, solar simulated ultraviolet irradiance increased the activity of XO and the NADPHdiaphorase reactivity as a protective response to formation of free radicals, such as reactive oxygen or nitrogen species. These pro-oxidant enzymes diminished in hypothyroid rats. Accumulation of the same amount of free radicals led to similar peroxidation in both hypothyroid and irradiated euthyroid rats. Hypothyroid skin after UV-exposure showed even greater lipid peroxidation.

Discussion: The hypothyroid state could be a risk factor for enhanced oxidative skin damage in chronic photo-exposed skin due to oxidative stress. The lipid peroxidation is one of the major pathways by which photo-oxidative stress promotes photocarcinogenesis and photo-aging.     

The effect of thymus extracts on phosphorus compounds in muscle and serum, and on serum calcium

1. Thymectomy in young rabbits decreased the ATP content and increased the inorganic phosphate content of skeletal muscle. The serum calcium content was decreased, whereas the inorganic phosphate content was increased. 2. The administration of a lipid fraction (TL) or protein fractions (CIF and TP) of thymus extracts to thymectomized rabbits in short-term experiments increased the ATP content of muscle and decreased the inorganic phosphate contents of muscle and serum. Serum calcium content was increased. 3. The action of the thymus extract TP was specific only on the phosphate compounds, since the increase in serum calcium concentration was also caused by the control extract from muscle. The action of the extract TL is not specific, being paralleled by the action of a control extract from muscle.     

Insulin Stimulates Translocation of Human GLUT4 to the Membrane in Fat Bodies of Transgenic Drosophila melanogaster

The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in ‘diabetic’ flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.