The structures of barium hexafluorosilicate and cesium hexafluororhenate(V)

The crystal structure of CsReF₆, together with a reinvestigation of that of BaSiF₆, is reported. Both have been determined from single crystal three-dimensional X-ray diffraction data. The structure of BaSiF₆ has been found to conform to the initially assigned space group R$\text{3}$m, contrary to the suggestions of other workers. The unit cell of BaSiF₆ has the dimensiona a_hex 7.189(1), c_hex 7.015(1) Å; Z = 3. Refinement by a least squares method gave R 0.0079 and R_w 0.0077. Crystals of CsReF₆ belong to the lower symmetry rhombohedral space group R$\text{3}$. The unit cell has the dimensions a_hex 7.853(1), c_hex 8.140(1) Å; Z = 3. Refinement gave R 0.031 and R_w 0.030. The lowering of symmetry is caused by rotation of the ReF₆^? octahedra about the 3-fold axis through each Re atom, causing CsReF₆ to have the KOsF₆ structure.     

The topography of soft,adhesive diatom ‘trails’ as observed by Atomic Force Microscopy

Gliding diatoms foul surfaces by leaving behind ‘trails’ of secreted mucilage. Atomic force microscopy (AFM) used in ‘fluid tapping’ mode enabled the topography of the soft, adhesive trails in the natural hydrated state to be imaged, and without the artefacts resulting from fixation and/or dehydration. Diatom trails consist of a continuous, swollen ridge of material that dominates the trail, as well as a diffuse hydrated mucilage coating observed on either side of the main trail. The main trail material is evenly attached to the coverslip along its entire length, and appears to cure, or become less soft/adhesive, over time. Diatom trails observed with the scanning electron microscope were severely damaged by dehydration, while trails imaged by the AFM in ‘contact’ mode were damaged and/or removed by the action of the cantilever. The AFM used in ‘fluid tapping’ mode is an excellent tool for topographical studies of soft/adhesive biological molecules in the hydrated state, and will have great value for measuring their physical and mechanical properties when operated in ‘force modulation’ mode.     

VARIATIONS IN THE SUBSTITUTED 3‐LINKED MANNANS CLOSELY ASSOCIATED WITH THE SILICIFIED WALLS OF DIATOMS1

The polysaccharides from cleaned frustules of the diatoms Pinnularia viridis (Nitzsch) Ehrenberg, Craspedostauros australis Cox, Thalassiosira pseudonana Hasle et Heimdal, and Nitzschia navis‐varingica Lundholm et Moestrup were extracted with hot alkali that dissolved the silica and were characterized by constituent sugar and linkage analyses. The polysaccharides from P. viridis were investigated further by permethylation, partitioning according to solubility, desulfation, and CD₃I‐methylation. Yields of carbohydrate in the hot alkali extracts ranged from 0.9% to 1.8% w/w based on the dry weight of the silica. Mannose was the dominant sugar in the polysaccharides from all four species (54–69 mol% of constituent sugars), although 14 other monosaccharides, including neutral sugars (glucose, galactose, xylose, arabinose, rhamnose, fucose), acidic sugars (glucuronic acid, galacturonic acid, 2‐O‐methylglucuronic acid), and O‐methylated neutral sugars (2‐O‐methylrhamnose, 3‐O‐methylrhamnose, 2,3‐di‐O‐methylrhamnose, 3‐O‐methylxylose, 4‐O‐methylxylose) were also detected in varying proportions among the four samples. The polysaccharides were predominantly composed of a 3‐linked mannopyranose backbone with a prevalence of linkage and/or substitution at O‐2 of the 3‐linked mannopyranosyl residues, and they were polyanionic, bearing uronic acid residues and/or sulfate esters. There were, however, species‐specific differences in the degree and position of substitution on the mannan backbone, the type and substitution patterns of the anionic substituents, and the type and linkage patterns of sugars other than mannose. Although definitive functions for these polysaccharides in diatom biology remain uncertain, a possible role in biosilicification is discussed.     

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1: implications for prostate cancer

In humans, thromboxane (TX) A(2) signals through the TPα and TPβ isoforms of the TXA(2) receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPβ involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1(640-942)). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPβ did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPβ led to phosphorylation of histone H3 at Thr(11) (H3 Thr(11)), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPβ in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPβ-mediated H3 Thr(11) phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA(2) as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPβ, and leading to H3 Thr(11) phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.     

Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs)

The synthesis and inhibitory activity of a series of 5-substituted-(1,1-dioxo-2,3-dihydro-1H-1 lambda(6)-benzo[e][1,2]thiazin-4-ylidene)-thiazolidine-2,4-dione derivatives as competitive inhibitors of recombinant bacterial arylamine-N-acetyltransferases (NATs) are described. The most potent NAT inhibitors are those that contain planar hydrophobic substituents on the sultam nitrogen.     

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

A new technique for preparing biominerals for atomic-force microscopy

Summary We describe a methodology that enables the orienting and cleaving of biominerals to consistently yield low z-axis (height) surfaces suitable for high-resolution imaging by atomic-force microscopy. The methodology permits biominerals to be prepared without chemical fixation, resin embedding, or sectioning, and demonstrates a utility for a variety of silicified and calcified biominerals.     

Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

K_ATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P₂ and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing K_ATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue ‘Evolution brings Ca²⁺ and ATP together to control life and death’.