The Implication of Early Chromatin Changes in X Chromosome Inactivation

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Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.     

Homology modeling and substrate binding study of human CYP4A11 enzyme

Although both bacterial CYP102 (P450BM3) and mammalian CYP4A isozymes share a common function as fatty acid hydroxylases, distinctly different preferred sites of oxidation are observed with the CYP102 performing the usual non-terminal hydroxylation or epoxidation and the CYP4A enzymes performing the unusual and enigmatic terminal hydroxylation. The origin of this unique product specificity in human CYP4A11 has been explored in this work, focusing on possible differences in the binding site architecture of the two isozymes as the cause. To this end, 3D model structures of the human CYP4A11 enzyme were built and compared to the X-ray structure of CYP102. The substrate-binding channel identified in CYP4A11 was found to have a much more sterically restricted active site than that in CYP102 that could cause limited access of long-chain fatty acid to the ferryl oxygen leading to the preferred omega-hydroxylation. Results of docking of a common substrate, lauric acid, into the binding site of both CYP4A11 and CYP102 and molecular dynamics simulations provided additional support for this hypothesis. Specifically, in the CYP4A11-lauric acid simulations, the omega hydrogens were closest to the ferryl oxygen most of the time. By contrast, in the CYP102-lauric acid complex, the substrate could penetrate further into the active site providing access of the non-terminal (omega-1, omega-2) positions to the ferryl oxygen. These results, taken together, have elucidated the origin of the unusual product specificity of CYP4A11 and illustrated the central role of binding site architecture in subtle modulation of function.     

Potentials and limitations of histone repeat sequences for phylogenetic reconstruction of Sophophora.

Simplified DNA sequence acquisition has provided many new data sets that are useful for phylogenetic reconstruction, including single- and multiple-copy nuclear and organellar genes. Although transcribed regions receive much attention, nontranscribed regions have recently been added to the repertoire of sequences suitable for phylogenetic studies, especially for closely related taxa. We evaluated the efficacy of a small portion of the histone repeat for phylogenetic reconstruction among Drosophila species. Histone repeats in invertebrates offer distinct advantages similar to those of widely used ribosomal repeats. First, the units are tandemly repeated and undergo concerted evolution. Second, histone repeats include both highly conserved coding and variable intergenic regions. This composition facilitates application of "universal" primers spanning potentially informative sites. We examined a small region of the histone repeat, including the intergenic spacer segments of coding regions from the divergently transcribed H2A and H2B histone genes. The spacer (about 230 bp) exists as a mosaic with highly conserved functional motifs interspersed with rapidly diverging regions; the former aid in alignment of the spacer. There are no ambiguities in alignment of coding regions. Coding and noncoding regions were analyzed together and separately for phylogenetic information. Parsimony, distance, and maximum-likelihood methods successfully retrieve the corroborated phylogeny for the taxa examined. This study demonstrates the resolving power of a small histone region which may now be added to the growing collection of phylogenetically useful DNA sequences.     

Morphological control of inositol-1,4,5-trisphosphate-dependent signals

Inositol-1,4,5-trisphosphate (InsP(3))-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP(3) itself is not generally available. In particular, it has not been determined how the interplay of InsP(3) generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP(3) signaling. Here, we explore the spatial and temporal characteristics of [InsP(3)](cyt) during a bradykinin-induced calcium wave in a neuroblastoma cell. This is achieved by using a unique image-based computer modeling system, Virtual Cell, to integrate experimental data on the rates and spatial distributions of the key molecular components of the process. We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP(3)](cyt) in the neurite. This requisite InsP(3) spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane. In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.     

Phylogenetic relationships in Nuphar (Nymphaeaceae): evidence from morphology,chloroplast DNA,and nuclear ribosomal DNA

The genus Nuphar consists of yellow-flowered waterlilies and is widely distributed in north-temperate bodies of water. Despite regular taxonomic evaluation of these plants, no explicit phylogenetic hypotheses have been proposed for the genus. We investigated phylogenetic relationships in Nuphar using morphology and sequences of the chloroplast gene matK and of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Two major lineages within Nuphar are consistently resolved with the morphological and molecular data sets. One lineage comprises New World taxa and the other represents a primarily Old World lineage. Relationships within the major lineages were poorly resolved by morphology and ITS, yet certain relationships were elucidated by all analyses. Most notable is the strong support for a monophyletic lineage of dwarf taxa and the alliance of the North American N. microphylla with the Eurasian taxa. Minor discordance between the independent cladograms is accounted for by hybridization. The common taxonomic practice of uniting all North American and Eurasian taxa under one species is not supported phylogenetically.     

Effects of heavy metals on immunocompetence of white-footed mice (Peromyscus leucopus)

Continued human population growth and industrialization result in increased contamination of wildlife habitats. Effects of such habitat deterioration on the well-being of natural populations are unclear. Exposure to contaminants may impair immunocompetence, thereby increasing disease susceptibility. The mammalian immune system is important in maintaining health and in its sensitivity to toxins. In our study conducted from May 1999 through May 2001, we examined assays of immnnoompetence in the white-footed mouse (Peromyscus leucopus) that inhabited reference sites and sites significantly contaminated with mixtures of heavy metals. We estimated potential exposure and uptake of heavy metals by measuring the level of each contaminant in representative soil and tissue samples. Intraindividual variation across mice, but not sex, explained a large portion of the overall variance in immune response, and spleen weight was significanltly afflicted by mouse age. We found no evidence that residence on contaminated sites had any effect on immunopathology and humoral immunity as measured in our study. We suggest that field and laboratory studies in ecotoxicology provide estimates of exposure to contaminants (i.e., tissue analyses) to establish a database suitable to clarify the dose-response relationship between contaminants and target systems.     

The G glycoprotein of respiratory syncytial virus depresses respiratory rates through the CX3C motif and substance P

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease.