Bisquaternary caracurine V derivatives as allosteric modulators of ligand binding to M2 acetylcholine receptors

The allosteric effect on muscarinic acetylcholine M2 receptors of 11 bisquaternary salts of the Strychnos alkaloid caracurine V was determined. The effect was indicated by the concentration which retarded the rate of dissociation of the antagonist [3H]-N-methylscopolamine from porcine cardiac cholinoceptors by a factor of 2 (EC50). The most potent compounds carry allyl and propargyl substituents, respectively. Introduction of more bulky substituents (e.g., benzyl groups) resulted in a considerably reduced allosteric potency. The wide range of EC50 values (3 nM for R = allyl. 1750 nM for R = 2-naphthyl) suggests a sterically restricted binding pocket. Molecular modeling studies indicated that the caracurine V ring system satisfies the pharmacophore model for the allosteric interaction.     

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.     

A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities

Background  

The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.     

Geochemically distinct carbon isotope distributions in Allochromatium vinosum DSM 180T grown photoautotrophically and photoheterotrophically

Anoxygenic, photosynthetic bacteria are common at redox boundaries. They are of interest in microbial ecology and geosciences through their role in linking the carbon, sulfur, and iron cycles, yet much remains unknown about how their flexible carbon metabolism—permitting either autotrophic or heterotrophic growth—is recorded in the bulk sedimentary and lipid biomarker records. Here, we investigated patterns of carbon isotope fractionation in a model photosynthetic sulfur‐oxidizing bacterium, Allochromatium vinosum DSM180^T. In one treatment, A. vinosum was grown with CO₂ as the sole carbon source, while in a second treatment, it was grown on acetate. Different intracellular isotope patterns were observed for fatty acids, phytol, individual amino acids, intact proteins, and total RNA between the two experiments. Photoautotrophic CO₂ fixation yielded typical isotopic ordering for the lipid biomarkers: δ¹³C values of phytol > n‐alkyl lipids. In contrast, growth on acetate greatly suppressed intracellular isotopic heterogeneity across all molecular classes, except for a marked ¹³C‐depletion in phytol. This caused isotopic “inversion” in the lipids (δ¹³C values of phytol < n‐alkyl lipids). The finding suggests that inverse δ¹³C patterns of n‐alkanes and pristane/phytane in the geologic record may be at least in part a signal for photoheterotrophy. In both experimental scenarios, the relative isotope distributions could be predicted from an isotope flux‐balance model, demonstrating that microbial carbon metabolisms can be interrogated by combining compound‐specific stable isotope analysis with metabolic modeling. Isotopic differences among molecular classes may be a means of fingerprinting microbial carbon metabolism, both in the modern environment and the geologic record.     

Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP

The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of development is incomplete. We present here a detailed expression profile of this promoter investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development, and in all the vegetative and floral cell and tissue types. GFP expression was not detected during the early stages of embryogenesis. The first perceptible GFP expression was observed in a small area at the junction of hypocotyl and cotyledons in embryos at around 13 days after anthesis. The GFP fluorescence progressively became stronger and expanded throughout the cotyledon and hypocotyl as embryo development advanced. After germination, varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen exhibited a low level of greenish autofluorescence, it was possible to discern GFP-dependent fluorescence in some of the pollen from all the T₀ plants examined. Developing cotton fibers also exhibited GFP fluorescence suggesting that the 35S promoter was active in these specialized epidermal cells. Thus, we show that the expression of the 35S promoter was developmentally regulated during embryogenesis and that beyond a certain stage during embryogenesis, the promoter was expressed in most cell and tissue types in cotton albeit at different levels.     

Estimating downwind concentrations of viable airborne microorganisms in dynamic atmospheric conditions.

A Gaussian plume model has been modified to include an airborne microbial survival term that is a best-fit function of laboratory experimental data of weather variables. The model has been included in an algorithm using microbial source strength and local hourly mean weather data to drive the model through a summer- and winter-day cycle. For illustrative purposes, a composite airborne "virus" (developed using actual characteristics from two viruses) was used to show how wind speed could have a major modulating effect on near-source viable concentrations. For example, at high wind speeds such as those occurring during the day, or with short travel times, near-source locations experience high viable concentrations because the microorganisms have not had time to become inactivated. As the travel time increases, because of slow wind speed or longer distances, die-off modulation by sunshine, relative humidity, temperature, etc., potentially becomes increasingly predominant.     

Engineering the stem cell microenvironment

Multipotent stem cells in the body facilitate tissue regeneration, growth, and wound healing throughout life. The microenvironment in which they reside provides signals that direct these progenitors to proliferate, differentiate, or remain dormant; these factors include soluble molecules, the extracellular matrix, neighboring cells, and physical stimuli. Recent advances in the culture of embryonic stem cells and adult progenitors necessitate an increased understanding of these phenomena. Here, we summarize the interactions between stem cells and their local environment, drawing on in vivo observations and tissue culture studies. In addition, we describe novel methods of characterizing the effects of various environmental factors and review new techniques that enable scientists and engineers to more effectively direct stem cell fate.