The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.

No abstract

We describe here a novel class of cis-acting response elements for retinoid, vitamin D, and estrogen receptors which are widely spaced (10 to 200 bp) direct repeats (DRs) of the canonical 5-AGGTCA half-site recognition motif (DR10 to DR200). In contrast to the specificity previously observed with shortly spaced DRs (DR1 to DR5), the different receptors bind promiscuously to these novel elements to activate transcription in the presence of retinoic acid (RA), vitamin D, or estrogen. The greatest RA-dependent transactivation, seen with DR15, was similar to that observed with the canonical DR5. Both RA receptors and retinoid X receptors contribute to transactivation through widely spaced DR elements. With the estrogen receptor, DR15 was one-third as efficient as the classical palindromic response element. A further increase of spacer lengths progressively decreased the efficiency of transactivation. No transactivation was seen with widely spaced DRs when the thyroid and retinoid X receptors were coexpressed in the presence of their ligands. The progesterone receptor was also unable to transactivate through a DR10 element composed of its cognate binding motifs. These results considerably extend the response element repertoire of nuclear receptors and suggest the existence of promiscuous transcriptional regulation through common response elements, as well as the possibility of receptor ''cross-talk.''The members of the superfamily of nuclear receptors for steroid/thyroid hormones, vitamin D, and retinoic acids (RAs) are known to act as ligand-inducible transcriptional trans regulators by binding to cognate cis-acting regulatory elements referred to as ligand response elements (for reviews, see references 4, 8, 14, 18-20, 22, 35, 39, and 58). In general, ligand response elements are composed of distinct arrangements of the core motif 5Ј-PuG[G/T][T/A]CA-3Ј or closely related sequences, but some families of receptors exhibit distinct nucleotide preferences for a given position. For example, all steroid receptors require a G in position 3, while RA receptors (RARs), retinoid X receptors (RXRs), and thyroid hormone receptors (TRs) have a preference for G over T at this position. In contrast to the estrogen receptor (ER), which requires a T at position 4, the members of the glucocorticoid receptor family (glucocorticoid, mineralocorticoid, progesterone, and androgen receptors) bind only to motifs containing an A at this position. Alterations in other positions can also result in differential recognition by the various receptors. Mutational and crystal structure analyses have shown that three amino acids in the so-called P-box of the first zinc finger in the DNA-binding domain (DBD) are critically involved in the recognition of the core motif (10, 41, 43, 52, 55; for recent reviews, see references 18 and 19).In addition to receptor-specific differences in the recognition of the precise sequence of the core motif, specific binding of receptors also results from distinct arrangements of the repeated motifs. Steroid receptors ...

No abstract

The innate immune systems of humans and other animals are activated by lipopolysaccharides (LPS), which are glucosamine-based phospholipids that form the outer leaflet of the outer membranes of Gram-negative bacteria. Activation involves interactions of LPS with the innate immunity-receptor comprised of toll-like receptor 4 in complex with so-called MD-2 protein and accessory proteins, such as CD14 and LPS binding protein. The Lipid Metabolites and Pathways Strategy (LIPID MAPS) Consortium has isolated in large amounts a nearly homogeneous LPS, Kdo(2)-Lipid A, and demonstrated that it activates macrophages via toll-like receptor 4. The active form of LPS, monomer or aggregate, is controversial. We have therefore examined the aggregation behavior and other physical properties of Kdo(2)-Lipid A. Differential scanning calorimetry of Kdo(2)-Lipid A suspensions revealed a gel-to-liquid crystalline phase transition at 36.4 degrees C (T(m)). The nominal critical aggregation concentration, determined by dynamic light scattering, was found to be 41.2 +/- 1.6 nM below the T(m) (25 degrees C), but only 8.1 +/- 0.3 nM above the T(m) (37 degrees C). The specific molecular volume of Kdo(2)-Lipid A, obtained by densitometry measurements was found to be 3159 +/- 71 A(3) at 25 degrees C, from which the number of molecules in each aggregate was estimated to be 5.8 x 10(5). The aggregation behavior of Kdo(2)-Lipid A in the presence of lipoprotein-deficient serum suggests that Re LPS monomers and multimers are the active units for the immune system in the CD14-dependent and -independent pathways, respectively.

A parylene nanpore was built into multichannel microfluidic devices and a single protein channel was reconstituted in a bilayer lipid membrane (BLM) on the pore. Although BLMs on micropores used for channel recordings are usually too unstable for the exchange of solutions between the upper and lower sides, the BLMs formed in our nano­pores were stable enough to permit solution exchange.

Sphingosine and sphingosine 1-phosphate (S1P) are sphingolipid metabolites that act as signaling messengers to activate or inhibit multiple downstream targets to regulate cell growth, differentiation, and apoptosis. The amphiphilic nature of these compounds leads to aggregation above their critical micelle concentrations (CMCs), which may be important for understanding lysosomal glycosphingolipid storage disorders. We investigated the aggregation of sphingosine and S1P over a comprehensive, physiologically relevant range of pH values, ionic strengths, and lipid concentrations by means of dynamic light scattering, titration, and NMR spectroscopy. The results resolve discrepancies in literature reports of CMC and pK(a) values. At physiological pH, the nominal CMCs of sphingosine and S1P are 0.99 +/- 0.12 microM (pH 7.4) and 14.35 +/- 0.08 microM (pH 7.2), respectively. We find that pH strongly affects the aggregation behavior of sphingosine by changing the ionic and hydrogen-bonding states; the nominal critical aggregation concentrations of protonated and deprotonated sphingosine are 1.71 +/- 0.24 microM and 0.70 +/- 0.02 microM, respectively. NMR measurements revealed that the NH3+-NH2 transition of sphingosine occurs at pH 6.6, and that there is a structural shift in sphingosine aggregates caused by a transition in the predominant hydrogen-bonding network from intramolecular to intermolecular that occurs between pH 6.7 and 9.9.