Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

DNA bridging and antibridging: a role for bacterial nucleoid-associated proteins in regulating the expression of laterally acquired genes

Horizontal DNA transfer plays a major role in the evolution of bacteria. It allows them to acquire new traits rapidly and these may confer fitness advantages as the bacteria compete with others in the environment. Historically, the mechanisms of horizontal DNA transfer, chiefly conjugation, transformation and transduction, have received a great deal of attention. Less attention has been focused on the regulatory problems that may accompany the acquisition of new genes by lateral routes. How are these genes integrated into the existing regulatory circuits of the cell? Does a process of 'plug-and-play' operate, or are the new genes silenced pending the evolution of regulatory mechanisms that make their expression not only safe but also beneficial to both the gene and its new host? Recent research shows that bacterial nucleoid-associated proteins such as H-NS, HU and Fis are important contributors to the processes of regulatory integration that accompany horizontal gene transfer. A key emerging theme is the antagonism that exists between the DNA–protein–DNA bridging activity of the H-NS repressor and the DNA-bending and DNA-wrapping activities of regulatory proteins that oppose H-NS.     

Co‐evolution of gametes of the Greater Bandicoot Rat,Bandicota indica – a murine rodent from South‐East Asia

Most Old World mice and rats, subfamily Murinae, have a spermatozoon with an apical hook, a long tail and, as seen typically in eutherian mammals, a bilaterally flattened head. Dramatically different from this are the sperm of the Greater Bandicoot Rat, Bandicota indica. Here, we ask the question has the structure of the sperm head co‐evolved with that of the egg coat, the zona pellucida? For this, we first summarise the morphological features of the spermatozoon of B. indica that may relate to zona pellucida penetration at the time of fertilisation, and we confirm that the sperm head is generally round, not bilaterally flattened, in profile and has a huge acrosome. We then show that the zona pellucida around oocytes in tertiary follicles also differs from that of the other murine rodents in being only about 4 μm thick and, as demonstrated by lectin staining, has an unusual abundance of alpha‐L‐fucose. These findings indicate that both the male and female gametes of this South‐East Asian murine rodent are highly divergent in their structural organisation. One of the functional implications of this probably relates to sperm–zona interactions and the release of acrosomal enzymes that probably facilitate penetration by digestion of the zona matrix at the time of fertilisation.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

The role of insulator elements in large-scale chromatin structure in interphase

Insulator elements can be classified as enhancer-blocking or barrier insulators depending on whether they interfere with enhancer-promoter interactions or act as barriers against the spreading of heterochromatin. The former class may exert its function at least in part by attaching the chromatin fiber to a nuclear substrate such as the nuclear matrix, resulting in the formation of chromatin loops. The latter class functions by recruiting histone-modifying enzymes, although some barrier insulators have also been shown to create chromatin loops. These loops may correspond to functional nuclear domains containing clusters of co-expressed genes. Thus, insulators may determine specific patterns of nuclear organization that are important in establishing specific programs of gene expression during cell differentiation and development.     

DNA supercoiling is differentially regulated by environmental factors and FIS in Escherichia coli and Salmonella enterica

Although Escherichia coli and Salmonella enterica inhabit similar niches and employ similar genetic regulatory programmes, we find that they differ significantly in their DNA supercoiling responses to environmental and antibiotic challenges. Whereas E. coli demonstrates large dynamic transitions in supercoiling in response to growth phase, osmotic pressure and novobiocin treatment, supercoiling levels are much less variable in S. enterica. The FIS protein is a global regulator of supercoiling in E. coli, but it was found to have less influence over supercoiling control in S. enterica. These inter-species differences fine-tune gene promoters to endogenous supercoiling and FIS levels. Transferring a Salmonella virulence gene promoter (P(ssrA) ) into a new enteric host (E. coli) caused aberrant expression in response to stimulatory signals. Reciprocal horizontal transfer of topA promoters, which control expression of topoisomerase I, between E. coli and S. enterica revealed how these orthologous promoters have evolved to respond differentially to FIS and supercoiling levels in their cognate species. This also identified a previously unrecognized osmoregulation of topA expression that is independent of FIS and supercoiling in both E. coli and S. enterica. These findings suggest that E. coli and S. enterica may be unexpectedly divergent in their global regulation of cellular physiology.     

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.     

Ionic permeation free energy in gramicidin: a semimicroscopic perspective

Ion permeation through the gramicidin channel is studied using a model that circumvents two major difficulties inherent to standard simulational methods. It exploits the timescale separation between electronic and structural contributions to dielectric stabilization, accounting for the influence of electronic polarization by embedding the channel in a dielectric milieu that describes this polarization in a mean sense. The explicit mobile moieties are the ion, multipolar waters, and the carbonyls and amides of the peptide backbone. The model treats the influence of aromatic residues and the membrane dipole potential. A new electrical geometry is introduced that treats long-range electrostatics exactly and avoids problems related to periodic boundary conditions. It permits the translocating ion to make a seamless transition from nearby electrolyte to the channel interior. Other degrees of freedom (more distant bulk electrolyte and nonpolar lipid) are treated as dielectric continua. Reasonable permeation free energy profiles are obtained for potassium, rubidium, and cesium; binding wells are shallow and the central barrier is small. Estimated cationic single-channel conductances are smaller than experiment, but only by factors between 2 (rubidium) and 50 (potassium). When applied to chloride the internal barrier is large, with a corresponding miniscule single-channel conductance. The estimated relative single-channel conductances of gramicidin A, B, and C agree well with experiment.