Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and kcat, while Km− was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity

The Aromatic Features in Very Faint Dwarf Galaxies

We present optical and mid-infrared photometry of a statistically complete sample of 29 very faint dwarf galaxies (M_r > -15 mag) selected from the SDSS spectroscopic sample and observed in the mid-infrared with Spitzer IRAC. This sample contains nearby (redshift z<0.005) galaxies three magnitudes fainter than previously studied samples. We compare our sample with other star-forming galaxies that have been observed with both IRAC and SDSS. We examine the relationship of the infrared color, sensitive to PAH abundance, with star-formation rates, gas-phase metallicities and radiation hardness, all estimated from optical emission lines. Consistent with studies of more luminous dwarfs, we find that the very faint dwarf galaxies show much weaker PAH emission than more luminous galaxies with similar specific star-formation rates. Unlike more luminous galaxies, we find that the very faint dwarf galaxies show no significant dependence at all of PAH emission on star-formation rate, metallicity, or radiation hardness, despite the fact that the sample spans a significant range in all of these quantities. When the very faint dwarfs in our sample are compared with more luminous (M_r ~ -18 mag) dwarfs, we find that PAH emission depends on metallicity and radiation hardness. These two parameters are correlated; we look at the PAH-metallicity relation at fixed radiation hardness and the PAH-hardness relation at fixed metallicity. This test shows that the PAH emission in dwarf galaxies depends most directly on metallicity.Comment: submitted to Ap

Mapping far-IR emission from the central kiloparsec of NGC 1097

Using photometry of NGC 1097 from the Herschel PACS (Photodetector Array Camera and Spectrometer) instrument, we study the resolved properties of thermal dust continuum emission from a circumnuclear starburst ring with a radius ~ 900 pc. These observations are the first to resolve the structure of a circumnuclear ring at wavelengths that probe the peak (i.e. lambda ~ 100 micron) of the dust spectral energy distribution. The ring dominates the far-infrared (far-IR) emission from the galaxy - the high angular resolution of PACS allows us to isolate the ring's contribution and we find it is responsible for 75, 60 and 55% of the total flux of NGC 1097 at 70, 100 and 160 micron, respectively. We compare the far-IR structure of the ring to what is seen at other wavelengths and identify a sequence of far-IR bright knots that correspond to those seen in radio and mid-IR images. The mid- and far-IR band ratios in the ring vary by less than +/- 20% azimuthally, indicating modest variation in the radiation field heating the dust on ~ 600 pc scales. We explore various explanations for the azimuthal uniformity in the far-IR colors of the ring including a lack of well-defined age gradients in the young stellar cluster population, a dominant contribution to the far-IR emission from dust heated by older (> 10 Myr) stars and/or a quick smoothing of local enhancements in dust temperature due to the short orbital period of the ring. Finally, we improve previous limits on the far-IR flux from the inner ~ 600 pc of NGC 1097 by an order of magnitude, providing a better estimate of the total bolometric emission arising from the active galactic nucleus and its associated central starburst.Comment: Accepted for publication in the A&A Herschel Special Editio

A Study of Heating and Cooling of the ISM in NGC 1097 with Herschel-PACS and Spitzer-IRS

NGC 1097 is a nearby Seyfert 1 galaxy with a bright circumnuclear starburst ring, a strong large-scale bar and an active nucleus. We present a detailed study of the spatial variation of the far infrared (FIR) [CII]158um and [OI]63um lines and mid-infrared H2 emission lines as tracers of gas cooling, and of the polycyclic aromatic hydrocarbon (PAH) bands as tracers of the photoelectric heating, using Herschel-PACS, and Spitzer-IRS infrared spectral maps. We focus on the nucleus and the ring, and two star forming regions (Enuc N and Enuc S). We estimated a photoelectric gas heating efficiency ([CII]158um+[OI]63um)/PAH in the ring about 50% lower than in Enuc N and S. The average 11.3/7.7um PAH ratio is also lower in the ring, which may suggest a larger fraction of ionized PAHs, but no clear correlation with [CII]158{\mu}m/PAH(5.5 - 14um) is found. PAHs in the ring are responsible for a factor of two more [CII]158um and [OI]63um emission per unit mass than PAHs in the Enuc S. SED modeling indicates that at most 25% of the FIR power in the ring and Enuc S can come from high intensity photodissociation regions (PDRs), in which case G0 ~ 10^2.3 and nH ~ 10^3.5 cm^-3 in the ring. For these values of G0 and nH PDR models cannot reproduce the observed H2 emission. Much of the the H2 emission in the starburst ring could come from warm regions in the diffuse ISM that are heated by turbulent dissipation or shocks.Comment: 17 pages, 14 figures, 5 tables; accepted for publication in Ap

Peculiar Velocities into the Next Generation: Cosmological Parameters From Large Surveys without Bias from Nonlinear Structure

We investigate methods to best estimate the normalisation of the mass density fluctuation power spectrum (sigma_8) using peculiar velocity data from a survey like the Six degree Field Galaxy Velocity Survey (6dFGSv). We focus on two potential problems (i) biases from nonlinear growth of structure and (ii) the large number of velocities in the survey. Simulations of LambdaCDM-like models are used to test the methods. We calculate the likelihood from a full covariance matrix of velocities averaged in grid cells. This simultaneously reduces the number of data points and smooths out nonlinearities which tend to dominate on small scales. We show how the averaging can be taken into account in the predictions in a practical way, and show the effect of the choice of cell size. We find that a cell size can be chosen that significantly reduces the nonlinearities without significantly increasing the error bars on cosmological parameters. We compare our results with those from a principal components analysis following Watkins et al (2002) and Feldman et al (2003) to select a set of optimal moments constructed from linear combinations of the peculiar velocities that are least sensitive to the nonlinear scales. We conclude that averaging in grid cells performs equally well. We find that for a survey such as 6dFGSv we can estimate sigma_8 with less than 3% bias from nonlinearities. The expected error on sigma_8 after marginalising over Omega_m is approximately 16 percent.Comment: Accepted for publication in MNRAS, 12 pages, 5 figures. V2 Discussion clarified, 1 figure added, improvements to the text and figures; V3 Figure 5 Plotting error corrected, SN1a contours smalle

Tertiary structure of transfer ribonucleic acid

NRC publication: Ye

Mechanism of action of streptomycin : studies with polynucleotide phosphorylase and ribosomes

Some of the previous work on the mechanism of action of streptomycin has indicated the general area of nucleic acid metabolism as being a possible site of action. Specifically, a report has appeared that streptomycin inhibits the "exchange reaction" catalyzed by polynucleotide phosphorylase. Therefore, the possible action of streptomycin on polynucleotide phosphorylase from a streptomycin sensitive strain of Escherichia coli has been reinvestigated. In preparation for the study on polynucleotide phosphorylase, the nucleoside diphosphates of adenosine, guanosine, cytidine, and uridine were synthesized in sufficient yield and of a satisfactory purity by the recently developed method of Moffatt and Khorana. It was not possible to confirm the inhibition of the polynucleotide phosphorylase catalyzed "exchange reaction" reported by Kornberg, using enzyme from our streptomycin sensitive E. coli SA. Nor was it possible to demonstrate any inhibition of the polymerization reaction catalyzed by this enzyme, or any effect on the sedimentation properties of the polymer so formed. However, the diamines putrescine, cadaverine, spermine, and spermidine, while having no effect on the time course of the polymerization reaction, did lower the sedimentation coefficient of the polymer formed and cause a more heterogeneous polymer to be formed. Streptomycin has been suggested as an inhibitor of protein synthesis. Therefore, a study of the possible effects of streptomycin on ribosomes from E. coli was made. These subcellular particles have been shown to be a site of protein synthesis. Dihydrostreptomycin (DHSM) interacted strongly with the ribosomes. It was found that most of the ribonucleo-protein precipitated when ribosomes were dialyzed overnight at 4° against buffer containing about 500 μg./ml. of DHSM. A study of ultracentrifuge patterns of dialysates of ribosomes against lower levels of DHSM indicated that disruption, with a loss of discrete sedimentation coefficients, occurred when the level of DHSM was about 350 μg./ml. A study of elution patterns from DEAE-cellulose columns indicated only a partial change in the pattern after breakdown. Examination of sedimentation coefficients of ribosome dialysates at lower DHSM levels indicated no specific effect of DHSM. The autodegradation of ribosomes by polynucleotide phosphorylase was studied. DHSM, at low levels, had an effect on the time course of the auto-degradation. A study of the distribution of induced β-D-galactosidase associated with the ribosomes indicated that it was associated with that protein of the ribosomes not precipitated by DHSM. It can be concluded that streptomycin interacts strongly with ribosomes. This gives support to recent claims that streptomycin inhibits ‘in vitro' protein synthesis, and that the site of this inhibition is the ribosome.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Physical Studies on Glutamic Dehydrogenase

95 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1967.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Circular dichroism studies on the conformation of transfer ribonucleic acid in the presence of different divalent cations: Biochemistry

NRC publication: Ye