A Metagenomic Framework for the Study of Airborne Microbial Communities

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.     

Trace Metal Imaging of Sulfate-Reducing Bacteria and Methanogenic Archaea at Single-Cell Resolution by Synchrotron X-Ray Fluorescence Imaging

Metal cofactors are required for many enzymes in anaerobic microbial respiration. This study examined iron, cobalt, nickel, copper, and zinc in cellular and abiotic phases at the single-cell scale for a sulfate-reducing bacterium (Desulfococcus multivorans) and a methanogenic archaeon (Methanosarcina acetivorans) using synchrotron X-ray fluorescence microscopy. Relative abundances of cellular metals were also measured by inductively coupled plasma mass spectrometry. For both species, zinc and iron were consistently the most abundant cellular metals. M. acetivorans contained higher nickel and cobalt content than D. multivorans, likely due to elevated metal requirements for methylotrophic methanogenesis. Cocultures contained spheroid zinc sulfides and cobalt/copper sulfides.     

Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library

High-throughput fluorescent intercalator displacement (HT–FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT–FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.     

Laminin-induced Acetylcholine Receptor Clustering: An Alternative Pathway

The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1–induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK−/− mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.     

Potassium Fluxes in Chlamydomonas reinhardtii (I.Kinetics and Electrical Potentials)

Potassium influx and cellular [K+] were measured in the unicellular green alga Chlamydomonas reinhardtii after pretreatment in either 10 or 0 mM external K+ ([K]0). K+ (42K+ or 86Rb+) influx was mediated by a saturable, high-affinity transport system (HATS) at low [K+]0 and a linear, low-affinity transport system at high [K+]o. The HATS was typically more sensitive to metabolic inhibition (and darkness) than the low-affinity transport system. Membrane electrical potentials were determined by measuring the equilibrium distribution of tetraphenylphosphonium. These values, together with estimates of cytoplasmic [K+] (B. Malhotra and A.D.M. Glass [1995] Plant Physiol 108: 1537-1545), demonstrated that at 0.1 mM [K+]0 K+ uptake must be active. At higher [K+]0 (>0.3 mM) K+ influx appeared to be passive and possibly channel mediated. When cells were deprived of K+ for 24 h, the Vmax for the HATS increased from 50 x 10-6 to 85 x 10-6 nmol h-1 cell-1 and the Km value decreased from 0.25 to 0.162 mM. Meanwhile, cellular [K+] declined from 24 x 10-6 to 9 x 10-6 nmol cell-1. During this period influx increased exponentially, reaching its peak value after 18 h of K+ deprivation. This increase of K+ influx was not expressed when cells were exposed to inhibitors of protein synthesis. The use of 42K+ and 86Rb+ in parallel experiments demonstrated that Chlamydomonas discriminated in favor of K+ over Rb+, and this effect increased with the duration of K+ deprivation.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.     

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.     

The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme

A superoxide-forming oxidase from activated human neutrophil membranes was solubilized by two slightly different methods, then purified by "dye-affinity" chromatography. Kinetic studies of the purified preparations gave Vmax values of 5-10 mumol of O-2/min/mg of protein, and Km values for NADH and NADPH that were in reasonable agreement with values determined previously using particulate and crude solubilized preparations of the respiratory burst oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed prominent bands at 67, 48, and 32 kDa, together with some minor contaminants, whereas gel electrophoresis under non-denaturing conditions gave a single major band that when eluted and re-electrophoresed in the presence of sodium dodecyl sulfate showed bands at 67, 48, 32 kDa. We believe that all three bands represent oxidase components. The flavin content of the purified enzyme was 20.4 +/- 2.0 S.E. pmol of FAD/microgram of protein, whereas heme averaged 0.1 +/- 0.02 pmol/microgram and ubiquinone could not be detected. Assuming that the enzyme is composed of one 67-kDa subunit, one 48-kDa subunit, and one 32-kDa subunit (i.e. that its molecular mass is approximately 150 kDa), it can be calculated to have a turnover number of 700-1500 min-1, in agreement with a value reported previously for oxidase in a particulate O-2-forming system (Cross, A. R., Parkinson, J. F., and Jones, O. T. G. (1985) Biochem. J. 226, 881-884), and to contain the following quantities of redox carriers (mol/mol): FAD, 3.0; heme, 0.015; ubiquinone, less than 0.06. It remains to be determined whether this preparation represents the complete respiratory burst oxidase or is only the pyridine nucleotide dehydrogenating component of a more complex enzyme.     

A Model for the Regulation of K Influx, and Tissue Potassium Concentrations by Negative Feedback Effects upon Plasmalemma Influx

By means of a modified Michaelis-Menten equation for K⁺ influx, which includes terms for root and external K⁺ concentrations (root [K⁺] and [K⁺]₀, respectively) it is possible to predict the manner in which short-term (perturbation) fluxes of K⁺ into roots of barley plants (Hordeum vulgare cv Fergus) vary with root [K⁺] and [K⁺]₀. Influx values derived from this equation were used to predict changes of root and shoot [K⁺] and K⁺ absorption rates (as functions of time and [K⁺]₀) from a knowledge of K⁺ efflux, relative growth rates of roots and shoots, and the partitioning of absorbed K⁺ between these organs. A microcomputer program was employed to model these changes in low-salt plants following transfer to solutions in which [K⁺]₀ was maintained at values ranging from 5 to 1000 millimoles per cubic meter. The model was operated on the basis of 10 minute absorption periods which provided data for continuous `updating' of tissue [K⁺]. The simulations were undertaken for periods corresponding to 30 days. During this time the model accurately predicted the manner in which K⁺ influx and root and shoot [K⁺] gradually approach values which are essentially independent of [K⁺]₀. The computer program was also used to predict the outcome of changing various external and internal parameters of the proposed regulatory system. The results of these simulations are discussed in the context of current models for negative feedback control of ion fluxes.