Comparison between synthetic nuclear localization signal peptides from the steroid/thyroid hormone receptors superfamily.

The main objective of the study is to demonstrate that short basic peptides from the steroid/thyroid hormone receptors superfamily act as Nuclear Localization Signals out of receptors context. Such synthesized peptides, chemically coupled to Bovine Serum Albumin, were shown to enable the corresponding BSA-conjugate to be transported to the nucleus. A second objective is to demonstrate the utility of viral cointernalization as a good method for rapid quantitation, comparison and competition in nuclear entry.     

Sensitivity of selected members of the family Halobacteriaceae to quinolone antimicrobial compounds

Many members of the Halobacteriaceae are inhibited by quinolone compounds, which inhibit type II DNA topoisomerase. Ciprofloxacin was the most potent inhibitor, followed by ofloxacin and norfloxacin. Ciprofloxacin concentrations between 25 and 60 μg/ml caused 50% inhibition of the growth of most Haloferax and Haloarcula species. Halobacterium species were less sensitive. At sublethal concentrations, formation of elongated and/or swollen cells was observed in many species. The alkaliphilic Natronobacterium pharaonis was very sensitive (50% inhibition by ciprofloxacin, ofloxacin, and norfloxacin at concentrations between 4 and 15 μg/ml). The resistance of many members of the Halobacteriaceae to high concentrations of quinolone compounds may in part be due to the high magnesium concentrations present in the growth media. Haloferax volcanii was sensitive to 40 μg/ml ciprofloxacin when grown at suboptimal magnesium concentrations (0.1 M), but was hardly affected by 100 μg/ml of the inhibitor when grown in the presence of 0.5–0.75 M MgCl₂. It is suggested that the putative archaeal type II DNA topoisomerase has properties similar to those of the enzyme from Bacteria, although its sensitivity to quinolone antimicrobial compounds may be lower. Received: 6 November 1995 / Accepted: 26 February 1996     

Determinants of target gene specificity for steroid/thyroid hormone receptors

The molecular specificity of the receptors for steroid and thyroid hormones is achieved by their selective interaction with DNA binding sites referred to as hormone response elements (HREs). HREs can differ in primary nucleotide sequence as well as in the spacing of their dyadic half-sites. The target gene specificity of the glucocorticoid receptor can be converted to that of the estrogen receptor by changing three amino acids clustered in the first zinc finger. Remarkably, a single Gly to Glu change in this region produces a receptor that recognizes both glucocorticoid and estrogen response elements. Further replacement of five amino acids in the stem of the second zinc finger transforms the specificity to that of the thyroid hormone receptor. These findings localize structural determinants required for discrimination of HRE sequence and half-site spacing, respectively, and suggest a simple pathway for the coevolution of receptor DNA binding domains and hormone-responsive gene networks.     

Ligand-dependent synergy of thyroid hormone and retinoid X receptors.

The binding of thyroid hormone receptors to DNA is enhanced by heterodimerization with nuclear proteins. One such heterodimerization partner has recently been characterized as the retinoid X receptor. 9-cis-Retinoic acid has been identified as a natural ligand for retinoid X receptors, suggesting a potential receptor-mediated interaction between thyroid hormone and 9-cis-retinoic acid in the regulation of thyroid hormone-responsive genes. A transient cotransfection assay was used to test for such an interaction. When a complex thyroid hormone response element composed of both direct and inverted repeat hexamers was tested, these two ligands activated gene expression synergistically. In contrast, when the response element consisted only of directly repeated hexamers, unliganded retinoid X receptors enhanced thyroid hormone responsiveness, but 9-cis-retinoic acid induced no additional activation. The results suggest a unique mechanism to achieve differential suggest a unique mechanism to achieve differential thyroid hormone sensitivity of thyroid hormone-responsive genes within a cell. Genes with appropriate response elements will show amplification of the thyroid hormone response by 9-cis-retinoic acid in the presence of retinoid X receptors; other thyroid hormone-responsive genes will be influenced by retinoid X receptors, but not 9-cis-retinoic acid.     

Effects of retinoid and thyroid receptors during development of the inner ear

The sensory epithelia of the vertebrate inner ear are comprised of a complex pattern of hair cells and supporting cells. Several different families of signaling molecules have been shown to play a role in the development and maintenance of this structure. In particular, the steroid/thyroid receptor superfamily, and specifically retinoid and thyroid receptors appear to influence the determination, differentiation, maintenance, and possibly regeneration, of the sensory epithelia of the vertebrate inner ear. Clinical and experimental evidence demonstrates that changes in the relative availability of retinoic acid and thyroid hormone, the ligands for retinoid and thyroid receptors, result in perturbations in the development of hair cell sensory epithelia. Retinoic acid and retinoid receptors appear to play a role in early developmental events including cellular proliferation and determination whereas thyroid hormone and thyroid receptors appear to play a role in later events including differentiation and maintenance.     

Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells

Intracellular trafficking and localization of proteins can now be efficiently visualized by fusion of a polypeptide to the green fluorescent protein (GFP). Many spectral variants of this reagent are now available, providing powerful tools for studies in living cells. This approach is particularly useful for members of the steroid/nuclear receptor superfamily, since these molecules frequently undergo rapid subcellular redistribution on ligand activation. A major roadblock in the application of this technology concerns problems associated with transient transfections. This technique produces cell populations that are highly heterogeneous with respect to the newly introduced protein and usually contain the protein in a highly overexpressed state. In addition, long-term studies related to cell cycle and cellular differentiation are essentially impossible with this approach. These problems can be overcome by introduction of the GFP fusion into cells under appropriate induction control. We describe application of the tetracycline regulatory system to inducible control of a glucocorticoid receptor (GR)/GFP chimera. Intracellular concentrations of GFP-GR can be very effectively controlled in this system, providing an ideal environment in which to study subcellular trafficking of the receptor and interactions with a variety of intracellular targets.     

Expression of members of the Fgf family and their receptors during midfacial development

Members of the FGF family play diverse roles in patterning, cell proliferation and differentiation during embryogenesis. To begin to address their function during craniofacial development we have analyzed the expression of 18 members of the Fgf family (Fgf1-15, -17, -18 and -20) and the four members of the FGF-receptor family in the prospective midfacial region between E9.5 and E11.5 by whole-mount in situ hybridization. We show that at E9.5, Fgf3, -8, -9, -10 and -17 are broadly expressed in midfacial ectoderm. Concomitant with the outgrowth of the nasal processes at E10.5, expression of Fgf3, -8, -9, -10, -15, -17 and -18 was detected in spatially restricted regions of ectoderm at the edge of the nasal pit and at the oral edge of the medial nasal process. Expression of Fgf8, Fgf9, Fgf10 and Fgf17 was still observed in these domains at E11.5. In contrast to the restricted expression patterns of the ligands, FgfR1 and FgfR2 were broadly expressed in facial mesenchyme and ectoderm, respectively, indicating a wide competence of midfacial tissue to respond to FGF signaling.