Metal Ion Column Research Articles

The COS CGM Compendium. IV. Effects of Varying Ionization Backgrounds on Metallicity Determinations in the z < 1 Circumgalactic Medium

Metallicity estimates of circumgalactic gas based on absorption-line measurements typically require photoionization modeling to account for unseen ionization states. We explore the impact of uncertainties in the extreme-ultraviolet background (EUVB) radiation on such metallicity determinations for the z < 1 circumgalactic medium. In particular, we study how uncertainties in the power-law slope of the EUV radiation, α EUVB, from active galactic nuclei affect metallicity estimates in a sample of 34 absorbers with H i column densities between and measured metal ion column densities. We demonstrate the sensitivity of metallicity estimates to changes in the EUV power-law slope of active galactic nuclei, α EUVB, at low redshift (z < 1), showing that derived absorber metallicities increase on average by ≈0.3 dex as the EUV slope is hardened from α EUVB = −2.0 to −1.4. We use Markov Chain Monte Carlo sampling of photoionization models with α EUVB as a free parameter to derive metallicities for these absorbers. The current sample of absorbers does not provide a robust constraint on the slope α EUVB itself; we discuss how future analyses may provide stronger constraints. Marginalizing over the uncertainty in the slope of the background, we find that the average uncertainties in the metallicity determinations increase from 0.08 to 0.14 dex when switching from a fixed EUVB slope to one that freely varies. Thus, we demonstrate that EUVB uncertainties can be included in ionization models while still allowing for robust metallicity inferences.

Wolbachia translation initiation factor-1 is copiously expressed by the adult, microfilariae and infective larvae of Brugia malayi and competitively inhibited by tetracycline

The intracellular alphaproteobacteria, Wolbachia, is considered to be a future antimacrofilarial drug target as it is obligatory for filarial endurance. Characterizing wolbachial proteins is necessary to understand wolbachial mechanisms and also for discovering new drug entities. Translation initiation factor-1 (Tl IF-1) is an indispensable prokaryotic factor concerned with bacterial viability. This factor is prioritized as one of the most potent antibacterial drug target. To investigate its role in filarial biology, recombinant Wol Tl IF-1 was purified on metal ion column. The factor was found folded in its monomeric native conformation, and contained a buried fluorophore. Molecular modeling revealed that the factor belonged to the Oligomer Binding family, and consisted of the highly conserved S1 domain with 81.6% of the amino acids occupying the allowed regions in Ramachandran plot. In addition, Wol Tl IF-1 exhibited selective binding to the 30S ribosomal subunit, which declined progressively with tetracycline addition. Tetracycline perturbs interaction of Thr18 and Asn32 of the factor with ribosomal protein S4. The factor was immune-localized in adult, microfilariae (Mf) and infective larvae (L3) of Brugia malayi by immunoblotting. High expression was also observed in Wolbachia within B. malayi Mf, L3 and female adult parasite along the gravid uteri by the confocal microscopy. Therefore, Wol Tl IF-1 appears to be an essential Wolbachia factor whose inhibition leads to extensive cell apoptosis and premature killing of adult worms, validating the antifilarial potential of the factor.

Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi

The pleiotropic functions of disaccharide trehalose in the biology of nematodes and its absence from mammalian cells suggest that its biosynthesis may provide a useful target for developing novel nematicidal drugs. The trehalose-6-phosphate phosphatase (TPP), one of the enzymes of trehalose metabolism has not been characterized so far in nematodes except the free living nematode Caenorhabditis elegans where it's silencing results into lethal outcomes. This prompted us to clone and characterize Brugia malayi TPP in order to discover novel antifilarial drug target. The recombinant protein (Bm-TPP) was purified with apparent homogeneity on a metal ion column and it was found to possess high phosphatase activity with robust specificity for the substrate trehalose-6-phosphate. Bm-TPP was found to be a member of the HAD-like hydrolase super family II based on the conserved motifs required for catalytic reaction. The Km for substrate trehalose-6-phosphate was around 0.42mM with pH optimum ∼7.0 and the enzyme showed an almost absolute requirement for Mg2+ as a metal ion. Bm-TPP was expressed in all the life-stages of B. malayi. In the absence of an effective macrofilaricidal agent and validated antifilarial drug target, Bm-TPP bodes well as a rational drug target against lymphatic filariasis.

PARTIALLY COOLED SHOCKS: DETECTABLE PRECURSORS IN THE WARM/HOT INTERGALACTIC MEDIUM

I present computations of the integrated column densities produced in the post-shock cooling layers and in the radiative precursors of partially-cooled fast shocks as a function of the shock age. The results are applicable to the shock-heated warm/hot intergalactic medium (WHIM) which is expected to be a major baryonic reservoir, and contain a large fraction of the so-called "missing baryons". My computations indicate that readily observable amounts of intermediate and high ions, such as CIV, NV, and OVI are created in the precursors of young shocks, for which the shocked gas remains hot and difficult to observe. I suggest that such precursors may provide a way to identify and estimate the "missing" baryonic mass associated with the shocks. The absorption-line signatures predicted here may be used to construct ion-ratio diagrams, which will serve as diagnostics for the photoionized gas in the precursors. In my numerical models, the time-evolution of the shock structure, self-radiation, and associated metal-ion column densities are computed by a series of quasi-static models, each appropriate for a different shock age. The shock code used in this work calculates the nonequilibrium ionization and cooling, follows the radiative transfer of the shock self-radiation through the post-shock cooling layers, takes into account the resulting photoionization and heating rates, follows the dynamics of the cooling gas, and self-consistently computes the photoionization states in the precursor gas. I present a complete set of the age-dependent post-shock and precursor columns for all ionization states of the elements H, He, C, N, O, Ne, Mg, Si, S, and Fe, as functions of the shock velocity, gas metallicity, and magnetic field. I present my numerical results in convenient online tables.

METAL-ABSORPTION COLUMN DENSITIES IN FAST RADIATIVE SHOCKS

In this paper we present computations of the integrated metal-ion column densities produced in the post-shock cooling layers behind fast, radiative shock-waves. For this purpose, we have constructed a new shock code that calculates the non-equilibrium ionization and cooling; follows the radiative transfer of the shock self-radiation through the post-shock cooling layers; takes into account the resulting photoionization and heating rates; follows the dynamics of the cooling gas; and self-consistently computes the initial photoionization state of the precursor gas. We discuss the shock structure and emitted radiation, and study the dependence on the shock velocity, magnetic field, and gas metallicity. We present a complete set of integrated post-shock and precursor metal-ion column densities of all ionization stages of the elements H, He, C, N, O, Ne, Mg, Si, S, and Fe, for shocks with velocities of 600 and ~2000 km/s, corresponding to initial post-shock temperatures of 5e6 and 5e7 K, cooling down to 1000 K. We consider shocks in which the magnetic field is negligible (B=0) so that the cooling occurs at approximately constant pressure ("isobaric"), and shocks in which the magnetic pressure dominates everywhere such that the cooling occurs at constant density (isochoric). We present results for gas metallicities Z ranging from 1e-3 to twice the solar abundance of heavy elements, and we study how the observational signatures of fast radiative shocks depend on Z. We present our numerical results in convenient online figures and tables, available at http://wise-obs.tau.ac.il/~orlyg/shocks/

Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion Affinity Media and Direct Analysis by MALDI-TOF-MS: Figure 1.

INTRODUCTIONPhosphoproteins and peptides can be bound with high specificity to immobilized metal ions such as Fe(3+), Ni(2+), and Ga(3+). This technique can be used with either on-line or off-line MS analysis. However, elution of the phosphopeptides from the metal ion column prior to MS analysis can result in sample loss. Affinity-bound analytes, including phosphopeptides, can be directly analyzed by MALDI-MS without prior elution from the affinity media. Also, consecutive enzymatic reactions, such as phosphatase or carboxypeptidase Y digestion, can be carried out on affinity-bound peptides. When the affinity-bound phosphopeptides are treated with phosphatase, the number of phosphorylation sites can be determined based on the observation of 80-Da (or multiples of 80 Da) mass shifts in the MALDI-MS of the reaction mixture. Carboxypeptidase Y treatment of the affinity-bound phosphopeptides can also be used to cleave amino acids from the carboxyl terminus, with subsequent direct analysis of the enzymatic products by MALDI-MS to locate the phosphorylation sites on the bound phosphopeptides. This protocol details the preparation and use of Fe(3+) or Ga(3+) metal IMAC with the on-bead analysis of phosphopeptides by MALDI-MS. Enzymatic digestion of affinity-bound peptides is also described.

Purification, Cloning, Expression, and Properties of Mycobacterial Trehalose-phosphate Phosphatase

The trehalose-phosphate phosphatase (TPP) was purified from the cytosol of Mycobacterium smegmatis to near homogeneity using a variety of conventional steps to achieve a purification of about 1600-fold with a yield of active enzyme of about 1%. Based on gel filtration, the active enzyme had a molecular weight of about 27,000, and the most purified fraction also gave a major band on SDS-PAGE corresponding to a molecular weight of about 27,000. A number of peptides from the 27-kDa protein were sequenced and these sequences showed considerable homology to the trehalose-P phosphatase (otsB) of Escherichia coli. Based on these peptides, the M. smegmatis gene for TPP was cloned and expressed in E. coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. Most of the TPP activity in the crude E. coli sonicate was initially found in the membrane fraction, but it became solubilized in the presence of 0.2% Sarkosyl. The solubilized protein was purified to apparent homogeneity on a metal ion column and this fraction had high phosphatase activity that was completely specific for trehalose-P. The purified enzyme, either isolated from M. smegmatis, or expressed in E. coli, rapidly dephosphorylated trehalose-6-P, but had essentially no activity on any other sugar phosphates, or on p-nitrophenyl phosphate. The K(m) for trehalose-6-P was about 1.6 mm, and the pH optimum was about 7.5. The native enzyme showed an almost absolute requirement for Mg(2+) and was not very active with Mn(2+), whereas both of these cations were equally effective with the recombinant TPP. The enzyme activity was inhibited by the antibiotics, diumycin and moenomycin, but not by a number of other antibiotics or trehalose analogs. TPP activity was strongly inhibited by the detergents, Sarkosyl and deoxycholate, even at 0.025%, but it was not inhibited by Nonidet P-40, Triton X-100, or octyl glucoside, even at concentrations up to 0.3%. The purified enzyme was stable to heating at 60 degrees C for up to 6 min, but was slowly inactivated at 70 degrees C. Circular dichroism studies on recombinant TPP indicate that the secondary structure of this protein has considerable beta-pleated sheet and is very compact. TPP may play a key role in the biosynthesis of trehalose compounds, such as trehalose mycolates, and therefore may represent an excellent target site for chemotherapy against tuberculosis and other mycobacterial diseases.

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.

An analysis of HST UV spectra of Cyg OB2 stars

As a first step of a vigorous program to investigate the Wind Momentum - Luminosity Relationship (WLR) of Galactic O-stars by analyzing stars belonging to the same cluster we present UV HST observations of six supergiants and one giant in the Cyg OB2 association. Terminal and turbulent wind velocities, velocity laws and metal ion column densities are derived and mean ionization fractions are estimated. Turbulent velocities are mostly in the range 10-14% of $v_\infty$. The terminal velocities agree well with the average $v_\infty$ vs. spectral class relationship compiled by Kudritzki & Puls ([CITE]). We compare the observed $v_\infty$ vs. escape velocity (depending on the diagnostics of the stellar mass) correlation with the predictions of the radiatively driven wind theory and find better agreement with the spectroscopic masses rather than with the evolutionary ones. The β velocity field exponents are in the range 0.7-0.8, without any trend towards larger values. We show that for a single luminosity class there is a tight relationship between T eff and $ $ (the mean density at the point in the stellar wind, where half the wind terminal velocity is reached). In consequence, the ionization fractions show the same trend with both, T eff and $ $: we find that Nv increases with T eff , Siiv decreases and Civ does not clearly correlate. As a byproduct, we also derive interestellar Hi column densities towards Cyg OB2, which turn out to be quite large. For one object (Cyg OB2$\#$2) we find inconsistencies making the association membership questionable.

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu 2+ > Ni 2+ > Zn 2+. The iminodiacetic acid-Cu 2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of adsorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at around neutral pH. Considerable amounts of immobilized Zn 2+ and Ni 2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 m m) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.