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Enzymology| Volume 288, ISSUE 21, P14917-14925, May 24, 2013
Mechanism of Ferrous Iron Binding and Oxidation by Ferritin from a Pennate Diatom*
Stephanie Pfaffen
From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and
Raz Abdulqadir
the Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom
Nick E. Le Brun
Michael E.P. Murphy
To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
* This work was supported by a Natural Sciences and Engineering Research Council of Canada discovery grant and the Canadian Foundation for Innovation (to M. E. P. M.) and Biotechnology and Biological Sciences Research Council Grant BB/I021884/1 (to N. E. L. B.).
Open AccessPublished:April 02, 2013DOI:https://doi.org/10.1074/jbc.M113.454496
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A novel ferritin was recently found in Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65–2.2-Å resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.
Background: Ferritin stores iron by ferroxidation to form a mineral core, enabling diatom blooms upon iron input.
Results: Ferrous iron binds solely to ferroxidase site A anaerobically. Ferroxidation kinetics has two observed phases.
Conclusion: Ferrous iron and dioxygen binding to the di-iron ferroxidase site is stepwise.
Significance: Iron storage by ferritins requires a coordinated binding of iron and dioxygen.
Stopped-flow Spectroscopy
Ferritin is a ubiquitous, iron storage, and detoxifying protein found in mammals, plants, and many microorganisms. Ferritin is a 24-mer that forms a hollow sphere, takes up soluble ferrous iron, oxidizes it at di-iron ferroxidase centers, and stores the iron oxide mineral in its central cavity. Iron is subsequently released upon demand of the organism's metabolism (
Carrondo M.A.
Ferritins, iron uptake and storage from the bacterioferritin viewpoint.
Theil E.C.
Ferritins: dynamic management of biological iron and oxygen chemistry.
Acc. Chem. Res. 2005; 38: 167-175
). Mammalian ferritins are heteropolymers of two homologous monomers that can store thousands of iron atoms in the central cavity. The heavy (H)
The abbreviations used are: H, heavy; PmFTN, P. multiseries ferritin; EcBFR, E. coli bacterioferritin; EcFtnA, E. coli ferritin; TCEP, tris(2-carboxyethyl)phosphine; Fe (5), Fe (45), Fe (75), Fe (2 h), Fe (4 h), and Fe (o.n.) refer to the structures of PmFTN from crystals soaked in solutions of ferrous iron for 5 min, 45 min, 75 min, 2 h, 4 h, and overnight, respectively; Zn (1 h), refers to the structure of PmFTN from crystals soaked in Zn(II) for 1 h.
chain contains the ferroxidase site, whereas the light chain promotes iron core formation (
Hempstead P.D.
Yewdall S.J.
Fernie A.R.
Lawson D.M.
Artymiuk P.J.
Rice D.W.
Ford G.C.
Harrison P.M.
Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution.
). In contrast, bacterial ferritins are homopolymers in which the monomer contains both the ferroxidase site and the mineral nucleation sites. The homologous bacterioferritins additionally contain a heme group between two monomers that functions principally in iron release (
Yasmin S.
Andrews S.C.
Moore G.R.
Le Brun N.E.
A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral.
). Storage of iron is the main function of ferritins, and detoxification of iron and reactive oxygen species is a secondary protective function utilized under extreme oxidative stress (
Messerschmidt A. Poulos H.R. Weighardt K. Handbook of Metalloproteins. 2. Wiley, Chichester, UK2001: 771-781
). However, whether these roles constitute the primary function of bacterioferritins remains to be proven (
Recently, ferritin homologs were identified in five species of pennate diatoms but not in other stramenopiles using a PCR approach (
Marchetti A.
Parker M.S.
Moccia L.P.
Lin E.O.
Arrieta A.L.
Ribalet F.
Murphy M.E.
Maldonado M.T.
Armbrust E.V.
Ferritin used for iron storage in bloom-forming marine pennate diatoms.
). A phylogenetic analysis of diatom ferritin sequences with those from both prokaryotes and eukaryotes showed that diatom ferritins are clearly distinct from other eukaryotic ferritins but may be weakly associated with prokaryotic ferritins (
). Diatoms are unicellular photosynthetic organisms that play a major role in global primary production and carbon sequestration into the deep ocean (
Falkowski P.G.
Barber R.T.
Smetacek V.V.
Biogeochemical controls and feedbacks on ocean primary production.
). In many offshore areas of the open ocean, primary productivity and therefore CO2 uptake from the atmosphere is limited due to iron availability. These regions are sporadically pulsed with new iron inputs from dust or upwelling deep waters. Pennate diatoms readily bloom upon such iron additions and continue to grow and divide after iron levels return to a low and ambient level (
Martin J.H.
Fitzwater S.E.
Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic.
). The expression of ferritin is thought to facilitate the blooming of pennate diatoms after iron fertilization in the open ocean.
A crystal structure of recombinant, iron-soaked ferritin derived from the pennate diatom Pseudo-nitzschia multiseries (PmFTN) was resolved at 1.95-Å resolution (
). The structure confirmed the characteristic ferritin ferroxidase center, monomeric fold, and spherical assembly. Nevertheless, the ferroxidase center found in PmFTN shows key differences from those of other ferritins of known structure. Typical eukaryotic H chain ferritins have a di-iron ferroxidase center (
Bertini I.
Lalli D.
Mangani S.
Pozzi C.
Rosa C.
Turano P.
Structural insights into the ferroxidase site of ferritins from higher eukaryotes.
J. Am. Chem. Soc. 2012; 134: 6169-6176
); however, three iron atoms are observed in and around the PmFTN ferroxidase center: one is found in ferroxidase site B, and the other two are positioned toward the core. An unexpected finding was that the ferroxidase site A is occupied by a water molecule. The iron atom found at site B is coordinated by three glutamate residues (Glu-48, Glu-94, and Glu-130) conserved in all ferritins. A unique site C is found in PmFTN at which iron is coordinated by only one glutamate residue (Glu-44). A glutamate is found at position 44 only in diatoms and cyanobacteria, and moreover, no third iron site is found in human H chain ferritin or other eukaryotic ferritins.
To get a better understanding of the ferroxidase reaction and iron binding in PmFTN, we have determined the x-ray structures of several PmFTN crystals soaked for various durations in ferrous iron and zinc sulfate under aerobic and anaerobic conditions. Furthermore, stopped-flow kinetic analysis was applied to determine reaction phases of the ferroxidase reaction and to understand the iron oxidation mechanism in PmFTN.
Protein Expression and Purification
The construct used for protein expression was a pET28a(+) vector containing the coding region of PmFTN genomic DNA lacking the signal peptide and plastid-targeting sequences (
). The expressed protein is missing the proline at the N terminus compared with the sequence found at UniProt entry B6DMH6. Escherichia coli BL21(DE3) cells transformed with the expression vector were inoculated into 2× YT medium (16 g/liter Bacto Tryptone, 10 g/liter Bacto Yeast Extract, and 5 g/liter NaCl) and grown at 37 °C to an optical density of ∼0.8 at 600 nm. Protein expression was induced with addition of 0.2 mm isopropyl β-d-thiogalactosidase. The cells were incubated at 25 °C overnight and afterward pelleted by centrifugation. The pellet was resuspended in 20 mm Tris-HCl, pH 8, 1 mm TCEP, and 5% glycerol (v/v), and the cells were lysed using an EmulsiFlex-C5 homogenizer (Avestin). Insoluble cell debris was removed by centrifugation. The supernatant was treated with DNase I type 2 and filtered through a 0.8-μm syringe filter.
PmFTN was purified using a heat shock method as described by Marchetti et al. (
). Briefly, the cell extract was aliquoted in 1-ml fractions, heat-shocked for 5 min at 60 °C, and put on ice for 4–5 min. The precipitated E. coli proteins were removed by centrifugation, and the remaining supernatant was filtered through a 0.22-μm syringe filter. PmFTN was further purified using Source 15Q (GE Healthcare) resin. The buffer used for the Source 15Q purification was 20 mm Tris, pH 8 and 5% glycerol (v/v), and the salt gradient used was 0–50% 1 m NaCl. Purified PmFTN was dialyzed into 3% sodium dithionite (w/v), 1 m sodium acetate, pH 4.8, and 1 mm TCEP to remove bound iron to yield the apoprotein. Apo-PmFTN was further dialyzed into 50 mm MES, pH 6.5, 100 mm NaCl, and 1 mm TCEP (Buffer A). The cysteine residues were alkylated by first incubating PmFTN in Buffer A supplemented with 2 mm TCEP for 2 h at 37 °C with shaking followed by the addition of 10 mm iodoacetamide and incubation for 45 min at 37 °C with shaking.
Some early preparations retained minor DNA contamination, which did not prevent crystallization but prevented accurate determination of kinetic parameters. Thus, subsequent preparations used an alternative DNA precipitation by the addition of 10 μl of 10% polyethyleneimine (w/v) (
Lu Y.
Roe J.A.
Bender C.J.
Banci L.
Gralla E.B.
Valentine J.S.
New type 2 copper-cysteinate proteins. Copper site histidine-to-cysteine mutants of yeast copper-zinc superoxide dismutase.
Inorg. Chem. 1996; 35: 1692-1700
)/ml of supernatant instead of DNase I treatment. The reaction was gently shaken for 10 min on ice, and afterward, the DNA was pelleted by centrifugation. Also, 0.5 mm EDTA was included in the buffer used for Source 15Q chromatography.
PmFTN Crystallization and Structure Solution
Diffraction quality crystals of PmFTN grew by vapor diffusion in a 1:1 mixture of 20 mg/ml protein in Buffer A supplemented with 2 mm TCEP and 10 mm iodoacetamide with reservoir solutions of 0.1 m sodium acetate, pH 5.5, 1.1–1.4 m ammonium sulfate, and 0.9–1.4 m sodium chloride. The crystals were soaked in mother liquor supplemented with freshly prepared 2 mm ammonium ferrous sulfate hexahydrate for a time period of 5 min, 45 min, 4 h, or overnight or with 2 mm zinc sulfate for 1 h. The crystals were transferred to a cryoprotectant consisting of mother liquor supplemented with 30% glycerol (v/v) before flash freezing in liquid nitrogen.
To obtain crystal structures from anaerobic crystals, a PmFTN protein solution was brought into a glove box (Belle Technology UK) containing a dinitrogen atmosphere maintained at less than 35 ppm dioxygen. Anaerobic diffraction quality crystals of PmFTN grew in 0.1 m sodium acetate, pH 5.5, 1.3–1.5 m ammonium sulfate, and 0.9–1 m NaCl. The crystals were soaked anaerobically in mother liquor supplemented with 2 mm ammonium ferrous sulfate hexahydrate for time periods of 75 min and 2 h. Thereafter, crystals were transferred into mother liquor supplemented with 30% glycerol (v/v) and immersed anaerobically in liquid nitrogen through a specialized port in the glove box.
PmFTN data sets were collected at the Stanford Synchrotron Radiation Lightsource on beamline 7.1 at 1-Å wavelength. Data were processed using HKL2000 to resolutions of 1.65–2.2 Å. Phases were determined using MOLREP (
Vagin A.
Teplyakov A.
MOLREP: an automated program for molecular replacement.
J. Appl. Crystallogr. 1997; 30: 1022-1025
) with a previously determined PmFTN crystal structure (Protein Data Bank code 3E6S) as the search model after removal of the iron and solvent atoms (
). The initial model was edited in Coot (
Emsley P.
Cowtan K.
Coot: model-building tools for molecular graphics.
Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132
) and refined with Refmac5 (
Murshudov G.N.
Vagin A.A.
Dodson E.J.
Refinement of macromolecular structures by the maximum-likelihood method.
Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255
). Waters were added by running Coot Find Waters in Refmac5. Anomalous dispersion data were used to identify metal sites. Anomalous maps were obtained with the program fft using the model phases. Final occupancies for metal sites were set such that the B-factors were similar to that of the coordinating residues. Data collection and refinement statistics are shown in Table 1. Figures were generated with PyMOL.
TABLE 1Data collection and refinement statistics
Anaerobic Fe (75)
Anaerobic Fe (2 h)
Fe (4 h)
Fe (o.n.)
Zn (1 h)
Resolution range (Å) 48.52–2.00 (2.00–2.05) 48.4–2.10 (2.10–2.15) 42.55–2.10 (2.10–2.16) 41.36–1.95 (1.95–2.00) 42.59–2.20 (2.20–2.26) 47.95–1.65 (1.65–1.69) 42.51–2.00 (2.00–2.05)
Space group P23 P23 P23 P23 P23 P4212 P23
Unit cell dimensions (Å) a = b = c = 174.77 a = b = c = 174.32 a = b = c = 175.28 a = b = c = 175.32 a = b = c = 175.45 a = b = 126.25 c = 170.30 a = b = c = 175.11
No. subunits in ASU 8 8 8 8 8 6 8
Unique reflections 119,135 102,117 103,862 128,778 90,434 163,306 117,223
Completeness (%) 99.8 99.5 99.6 99.2 99.7 99.2 97.5
Redundancy 14.3 14.6 13.0 11.6 14.0 10.7 14.7
Average I/σI 23.8 (4.8) 32.3 (5.4) 35.5 (7.1) 33.4 (3.8) 21.9 (4.7) 32.1 (3.7) 29.5 (7.4)
Rmerge 0.115 (0.658) 0.084 (0.597) 0.075 (0.371) 0.064 (0.528) 0.122 (0.553) 0.065 (0.507) 0.086 (0.452)
Wilson B (Å2) 29.8 30.3 24.2 24.8 26.2 22.4 26.9
Rwork (Rfree) 0.187 (0.235) 0.201 (0.247) 0.176 (0.225) 0.173 (0.213) 0.187 (0.242) 0.176 (0.205) 0.184 (0.232)
Average B (Å2) 31.5 32.9 27.9 28.5 28.3 25.5 30.1
No. water 853 524 821 1,006 538 872 912
No. iron 8 8 6 16 35 24 —
No. zinc — — — — — — 32
r.m.s.d. bond length (Å) 0.020 0.019 0.020 0.024 0.019 0.028 0.021
ESU (maximum likelihood; Å) 0.099 0.116 0.102 0.081 0.120 0.051 0.096
Ramachandran plot (%)
In most favorable 96.6 96.0 96.5 96.3 96.9 96.7 96.0
In allowed 3.4 4.0 3.6 3.7 3.1 3.3 4.0
Protein Data Bank code 4ITW 4IXK 4ITT 4IWJ 4ISP 4IWK 4ISM
Ferroxidase Kinetics
Rapid kinetic experiments were carried out using a stopped-flow instrument (Applied Photophysics DX17MV). Changes in absorption on addition of ferrous iron to aerobic apo-PmFTN were measured. A 50 mm ferrous iron stock solution was prepared by dissolving ferrous ammonium sulfate in deoxygenated water, which was bubbled with argon gas for 2 h prior to use. The stock solution was acidified with 37% HCl. 10–400 μm ferrous iron working solutions were freshly prepared prior to each experimental run using the 50 mm stock solution. 1 μm apo-PmFTN in 100 mm MES, pH 6.5 and 200 mm NaCl was mixed (1:1) by the stopped-flow instrument with the various ferrous iron working solutions, resulting in a protein concentration of 0.5 μm during data acquisition. All stopped-flow experiments were carried out at 25 °C. Resulting data were fit to a single exponential function using the program Origin (v8, OriginLab). Regeneration experiments were carried out using 1 μm PmFTN containing 48 iron ions added under aerobic conditions for a fixed period (30 min or 20 h) prior to the stopped-flow experiment.
PmFTN Overall Fold
Protein crystals of recombinant PmFTN soaked in Fe(II) or Zn(II) under various conditions diffracted to at least 2.2-Å resolution. Analysis of Ramachandran plots showed that in all structures more than 96% of the residues were in the preferred regions. All but one of the crystals analyzed were of space group P23 with eight ferritin monomers in the asymmetric unit (Table 1). The refined structures are nearly complete with at most 12 residues absent from the N or C terminus. As shown previously, the structures confirm the typical ferritin arrangement (Fig. 1): 24 subunits assemble to form a hollow spherical shell (
). The monomers adopt a four-helix bundle plus a shorter C-terminal α-helix. No significant changes in the fold of the ferritin monomer were observed upon metal treatment. Superposition of the monomers from each structure resulted in a 0.28-Å root mean square deviation for all Cα atoms.
FIGURE 1Crystal structure of PmFTN. A, crystal structure of the recombinant iron-soaked P. multiseries ferritin multimer shows the typical ferritin arrangement in which the 24 subunits form a spherical shell. B, monomer showing the ferroxidase site side chains and bound iron atoms. Magenta, A helix; green, B helix; blue, C helix; yellow, D helix; red, E helix; orange spheres, iron atoms.
Iron Binding to PmFTN in the Absence of Dioxygen
The first step in the ferritin reaction is binding of ferrous iron to the ferroxidase site. To observe the binding of ferrous iron, apo-PmFTN crystals were grown in an anaerobic environment, and the octahedral crystals were soaked in 2 mm ferrous iron. Two crystal structures were obtained with crystals soaked in ferrous iron for 75 min and 2 h to resolutions of 2 and 2.1 Å, respectively. The ferroxidase site of the higher resolution structure obtained from the crystal soaked for 75 min is shown in Fig. 2A. Ferrous iron ions are exclusively found in the ferroxidase sites of each monomer. Furthermore, ferrous iron is observed solely in site A (Fig. 2A) and is refined with an occupancy of ∼50% and B-factors similar to the coordinating residues. The iron in site A is coordinated in an approximate tetrahedral arrangement by the side chains of Glu-15 (∼2.2 Å) and Glu-48 (∼2.2 Å). His-51 forms a weaker interaction with ferrous iron (2.5–2.9 Å). The coordination sphere is completed by one or two solvent molecules (∼2.1 and ∼2.5 Å) depending on the monomer in the asymmetric unit. Ferroxidase site B is occupied by a solvent molecule.
FIGURE 2Ferroxidase centers of PmFTN. Monomer A of the asymmetric unit is shown. Iron and zinc atoms are drawn as orange and gray spheres, respectively, and cyan spheres represent water molecules. Side chains of selected residues are drawn in sticks with carbon, nitrogen, and oxygen atoms in the backbone color, blue, and red, respectively. Solid lines are metal ligand bonds, and dashed lines are selected hydrogen bonds. A, crystal soaked for 75 min in ferrous iron under anaerobic conditions. One iron atom is bound at ferroxidase site A at 50% occupancy, and site B is occupied by a water molecule. The black mesh represents the anomalous map contoured at 5 σ. B, crystal soaked overnight in ferrous sulfate under aerobic conditions. Three iron atoms are bound at sites A, B, and C, and one further iron atom was found beyond site C. C, crystal soaked for 1 h in zinc sulfate. Three zinc atoms are bound at the ferroxidase sites A, B, and C. D, an omit map contoured at 1 σ around site A and site B of PmFTN soaked overnight in ferrous sulfate is shown as a gray mesh.
Iron Binding to PmFTN in the Presence of Dioxygen
In our previous work, apo-PmFTN crystals were soaked for 10 min in ferrous sulfate in the presence of dioxygen (
). The resulting structure revealed iron bound at two sites: ferroxidase site B and a new site C nearby. Site A was occupied by solvent. To determine whether occupation of ferroxidase sites and site C is time-dependent, apo-PmFTN crystals were soaked for 5 min, 45 min, 4 h, and overnight in 2 mm ferrous sulfate. Structures of crystals exposed to ferrous iron for 4 h or longer revealed iron bound in all three sites as well as up to two additional sites at the inner surface of the ferritin sphere (Figs. 2B and 3A). The latter two sites may serve as the nucleation site for formation of the mineral core. The iron in site A is coordinated by the conserved residues Glu-15, Glu-48, and His-51. Coordination of the iron bound in site B is by 2–3 glutamate residues, depending on the subunit. A bridging oxygen atom is modeled between sites A and B and between sites B and C (Fig. 2B). A second solvent oxygen atom is coordinated to iron in site A. The bridging solvent molecule between sites A and B might be a bridging oxo group rather than a water molecule, building a diferric-oxo bridge as seen in other ferritins (
). Sample electron density for the overnight-soaked structure at the ferroxidase site including the bridging ligand is presented in Fig. 2D. In all subunits, the side chain carboxylate of Glu-48 is a bridging ligand to sites A and B, whereas Glu-94 coordinates to iron bound to site B only. Glu-130 adopts multiple conformations depending on the soaking time and is observed coordinated to iron atoms in site B and/or site C. In the overnight-soaked structure, Glu-130 is tilted toward the iron in site B, whereas in the 4-h-soaked structure, it is predominately coordinated to site C (Fig. 3, A and B). In addition to Glu-130, the site C iron is coordinated by Glu-47 and Glu-44.
FIGURE 3Residue Glu-130 exhibits flexible coordination to site B and site C iron. A, monomer A of PmFTN soaked in iron for 4 h. Glu-130 is coordinating to site C and may also interact with the site B iron atom. B, monomer B of PmFTN soaked in iron overnight. Glu-130 is coordinating to site B and may also interact with the site C iron atom. C, monomer C of PmFTN soaked in zinc for 1 h. Glu-130 is coordinating to the site B iron or site C iron atom. Molecular representations and the color scheme are the same as in Fig. 2. Bond lengths are in Å.
Iron occupancy of the sites A, B, and C varies depending on the incubation time of the crystals in ferrous iron in the presence of dioxygen (Table 2). Iron is observed in site A only after soaking for 45 min (Fe (45)), whereas after 5 min (Fe (5)), iron partially occupies site B in six of eight crystallographic subunits. In the other two subunits, a water molecule is modeled at the equivalent position. The distance between the iron atoms in sites A and B is ∼3.8 Å in Fe (45), which decreases to ∼3.6 Å upon overnight soak. In general, site C, which is situated between the ferroxidase site (sites A and B) and the mineral core, has the lowest overall iron occupancy (40–60%). Only in crystals incubated in ferrous iron for 4 h and longer was iron bound to site C. In the subunits with water bound to site C, the side chain of Glu-130 adopts an alternative rotamer such that it is directed away from the site and toward the core.
TABLE 2Range of iron and zinc ion occupancy observed in ferroxidase sites A, B, and C of PmFTN aerobically soaked crystals
Site A H2O Fe (75–90%) Fe (75–90%) Fe (85–95%) Zn (100%)
Site B Fe (35–50%) Fe (40–50%) Fe (75–80%) Fe (80–85%) Zn (85–90%)
Site C H2O Fe (50–60%) Fe (40–50%) Zn (60–75%)
Core metal atoms Fe (50–60%) Fe (30–35%)
Fe (30–50%)
PmFTN Binds Zn(II)
Zinc ions are competitive inhibitors of ferritin and bacterioferritin (
Yang X.
Thomson A.J.
Chasteen N.D.
The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin.
). Crystal structures of Zn(II)-inhibited ferritin confirm that this metal binds to the ferroxidase site and site C. The zinc ions are coordinated by the same residues as seen in the structures from aerobically iron-soaked crystals (Fig. 2C). Glu-130 is predominately coordinated to the zinc ion in site C, although in three of the monomers of the asymmetric unit, Glu-130 adopts two equal occupancy conformations, one that coordinates to the zinc ion in site B, and the other that coordinates to the zinc ion in site C (Fig. 3C). Furthermore, the zinc ion at site A is modeled as coordinated by two water molecules, one of which is bridging to site B. This arrangement of coordinated solvent is similar to that observed in the iron-soaked structures. In the case of iron, the modeled oxygen atom may be an oxo bridge rather than water. An overlay of the zinc structure with the iron structures showed that the zinc atom in site B is slightly shifted toward site A and that the zinc atom in site C is closer toward the core as compared with the iron.
Stopped-flow Absorbance Spectroscopy
Stopped-flow experiments were performed to monitor kinetics of iron oxidation after ferrous iron addition to apo-PmFTN. Absorption was measured at 340 nm, which reports on the oxidation of Fe(II) to Fe(III). Fig. 4A shows the absorption changes following the mixing of 1 μm apo-PmFTN with a variable amount of ferrous iron as a function of time. Fe(II) oxidation occurred extremely rapidly with completion for all additions within 0.5 s. For the addition of ∼50 Fe(II) ions per protein (two Fe(II) ions per subunit), the t½ was <50 ms, demonstrating a rate of reaction that is an order of magnitude faster than those measured for EcFtnA and human H ferritin under comparable conditions (
Treffry A.
Zhao Z.
Quail M.A.
Guest J.R.
Dinuclear center of ferritin: studies of iron binding and oxidation show differences in the two iron sites.
How the presence of three iron binding sites affects the iron storage function of the ferritin (EcFtnA) of Escherichia coli.
FEBS Lett. 1998; 432: 213-218
). Each trace fitted well to a single exponential function (Fig. 4A), giving an observed (pseudo-first order) rate constant, k0, for each addition. Fig. 4B shows a plot of k0 as a function of Fe(II) concentration. Remarkably, there is a linear relationship, demonstrating a first order dependence of the rate of oxidation on the concentration of Fe(II). This observation is significant because it indicates that Fe(II) binding, rather than Fe(II) oxidation, is the rate-determining step of the reaction, consistent with the unprecedented overall rate of Fe(II) oxidation. The slope of the line gives an apparent second order rate constant, k, of 2.54 ± 0.10 × 105 m−1 s−1.
FIGURE 4Kinetic analyses of Fe(II) oxidation catalyzed by PmFTN. A, stopped-flow measurements of ΔA340 nm as a function of time following additions of variable Fe(II) (as indicated) to apo-PmFTN. Each of the A340 nm traces were fit to a single exponential function (solid lines). B, plot of observed (pseudo-first order) rate constants obtained from fitting the data in A as a function of Fe(II) concentration. Standard errors (error bars) are shown, and a linear fit of the data is drawn. C, plot of total amplitude of absorbance changes at 340 nm at 0.5 s (data from A) as a function of Fe(II) added per PmFTN protein. The two clear phases are highlighted, intersecting at ∼50 Fe(II) ions per protein. D, stopped-flow A340 nm measurements as a function of time following addition of 100 Fe(II) ions per apo-PmFTN. The initial very rapid oxidation of 48 Fe(II) ions per protein is not captured well on this extended time scale, but the subsequent, much slower oxidation of Fe(II) in excess of that needed to saturate the ferroxidase center sites is observed clearly. E, stopped-flow measurement of A340 nm over an extended time period revealing small increases in absorbance following the rapid oxidation phase. The number of Fe(II) ions per apo-PmFTN is indicated. F, stopped-flow measurements of absorbance changes at 340 nm following the addition of 40 or 80 Fe(II) ions per protein to a sample of PmFTN previously treated with 48 Fe(II) ions per protein under aerobic conditions. The incubation time between Fe(II) additions was 30 min. For all experiments, PmFTN (1 μm) was in 0.1 m MES, pH 6.5.
A plot of ΔA340 nm at 0.5 s versus Fe(II) added per PmFTN complex (Fig. 4C) reveals that the rapid oxidation phase is saturated at ∼50 iron ions per protein or two per subunit. Such behavior is characteristic of ferritins in which the ferroxidase center is the initial site of rapid Fe(II) oxidation. Clearly, Fe(II) added in excess of that required to saturate the ferroxidase center is not oxidized at a rate close to that of the initial oxidation phase, and so we conclude that Fe(II) does not undergo rapid oxidation at the third site (site C). This is demonstrated in Fig. 4D, which shows changes in A340 nm over 1000 s following the addition of 100 Fe(II) ions per protein. Although the first ∼50 Fe(II) ions are oxidized extremely rapidly, the subsequent 50 are not; in fact, oxidation is not complete at 1000 s. Thus, the kinetic distinction between these phases is unusually clear.
The partial occupancy of iron in the ferroxidase sites A and B after prolonged exposure to Fe(II) under aerobic conditions (Table 2) indicates that iron is likely to be mobile following oxidation at the ferroxidase center. Thus, A340 nm was monitored over a longer time period (Fig. 4E). The data show that a small increase in absorption occurs during the few seconds following the rapid oxidation phase. This increase was observed at all levels of Fe(II) additions, including those that are substoichiometric, and so these changes are not due to oxidation of Fe(II) bound elsewhere. Thus, these absorption changes may be attributed to reorganization of iron in the ferroxidase center and possibly site C following the initial rapid oxidation.
Mobility of iron following oxidation of Fe(II) at the ferroxidase center could result in complete loss of Fe(III) from the center, leading to regeneration of the apo form. This form would be expected to exhibit the rapid oxidation phase observed above (Fig. 4A). To determine whether the rapid oxidation phase is recovered, PmFTN was loaded with 48 Fe(II) ions per 24-mer and then subsequently incubated for 30 min or 20 h. Then, a further aliquot of Fe(II) (either 40 or 80 Fe(II) ions per protein) was added, and changes in A340 nm were measured. The data (for 30 min; Fig. 4F) show that no rapid phase of Fe(II) oxidation is observed. Two distinct phases are observed, although it is not yet clear what these correspond to. Data for 20 h are similar (not shown). Clearly, there is no full regeneration of the apo form of PmFTN following oxidation of the initial addition of Fe(II).
Iron storage ferritins are the archetype of the ferritin-like superfamily of proteins, which includes the enzymes ribonucleotide reductase and methane monooxygenase. A common feature is a catalytic di-iron site where dioxygen is reduced. In ferritin, Fe(II) is the source of electrons, and the resulting oxidized Fe(III) is deposited in the mineral core. A key question in ferritin function is the mechanism of iron mobilization in and out of the ferroxidase site and the core. Iron was visualized in the ferroxidase site (sites A and B) and a third site C of PmFTN. The residues interacting with the iron in site A (Glu-15, Glu-48, and His-51) are conserved with those of all characterized eukaryotic and prokaryotic ferritins. In addition to Glu-48, Glu-94 coordinates iron in site B and is also conserved.
A third iron site is generally not associated with ferritin ferroxidase sites from vertebrates; however, a site C has been observed in some non-heme ferritins from bacteria and archaea, for example in E. coli (EcFtnA) (
Stillman T.J.
Hudson A.J.
The high-resolution x-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe3+ and Zn2+ derivatives.
), Pyrococcus furiosus (
Tatur J.
Hagen W.R.
Matias P.M.
Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus.
J. Biol. Inorg. Chem. 2007; 12: 615-630
), and Archaeoglobus fulgidus (
Johnson E.
Cascio D.
Sawaya M.R.
Gingery M.
Schröder I.
Crystal structures of a tetrahedral open pore ferritin from the hyperthermophilic archaeon Archaeoglobus fulgidus.
Structure. 2005; 13: 637-648
). Nevertheless, these sites differ from that of PmFTN in terms of the number and origin of glutamate residues. Of these residues, Glu-130 is in common, is observed to bridge the iron atom of sites B and C (Fig. 3), and is a conserved residue in prokaryotic ferritins. In the PmFTN structure soaked aerobically for more than 4 h, Glu-130 is a ligand to iron atoms in sites B and C. In contrast, in the earlier PmFTN structure soaked in iron for 10 min by Marchetti et al. (
), the iron in site C is coordinated by only Glu-44. In all known prokaryotic ferritins, Glu-44 is substituted by histidine. The equivalent His-46 in EcFtnA is proposed to orient Glu-130 so it can bind iron ions in sites B and C as well as gating the passage of the metal through these sites (
). Glu-44 and Glu-130 in PmFTN may have a similar function of gating the passage of iron from site B to site C.
As the aerobic iron soaking time increases from minutes to hours, iron is observed first in site B followed by site A and eventually occupies all three sites (Table 2). In contrast, only site A is occupied by Fe(II) in crystals of PmFTN under anaerobic conditions even though the crystals were exposed to 2 mm Fe(II) for over an hour (Fig. 2A). This observation is in contrast to short (∼1-min) aerobic Fe(II) soaking experiments with frog ferritin in which both sites A and B are occupied (
). Interestingly, the interiron distance in some subunits of frog ferritin is comparable with that observed with Cu(II) as a proxy for Fe(II) (∼4.3 Å). Longer exposure to ferrous iron results in a shortening of the di-iron interatomic iron distance to ∼3.1 Å. In PmFTN, iron occupancy at both sites is only observed after prolonged iron exposure, and the site A and B intermetal distance of less than 3.8 Å decreases only slightly with time to 3.6 Å, suggesting that in the structures where iron is bound to both sites it is in the Fe(III) state. Note that with the data presented here we are not able to directly determine the oxidation state of iron bound to the crystals under aerobic conditions, including that in site B in Fe (5). A single high affinity and two low affinity Fe(II) binding sites were identified in P. furiosus ferritin by calorimetry in the absence of dioxygen (
Honarmand Ebrahimi K.
Bill E.
Hagedoorn P.L.
The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.
Nat. Chem. Biol. 2012; 8: 941-948
). Site-directed mutagenesis was used to propose assignment of the high affinity site to site A, consistent with our anaerobic crystallographic observations in PmFTN.
Kinetic measurements of PmFTN iron oxidation revealed an extremely rapid initial oxidation phase involving the binding and oxidation of two ferrous iron. The first order dependence of the rate of ferroxidase center oxidation on the concentration of Fe(II) demonstrates a close link between binding and oxidation events such that they cannot be distinguished. Thus, oxidation occurs immediately upon Fe(II) binding to PmFTN, and the binding event can be viewed as the slow step of the reaction. This is in contrast to previous reports of ferritins in which binding and oxidation are considered to be kinetically distinct events. Measurement of Fe(II) binding kinetics is not generally straightforward, although it was possible for EcBFR because Fe(II) binding caused a perturbation of absorbance due to the heme groups. In that case, Fe(II) binding occurred on a much shorter time scale than the subsequent Fe(II) oxidation. Interestingly, Fe(II) binding to EcBFR occurred with a second order rate constant of 2.5 × 105 m−1 s−1 (at 30 °C) (
), a value similar to that measured here (at 25 °C) for PmFTN-catalyzed Fe(II) oxidation.
An oximetric assay previously showed that the ferroxidase reaction of PmFTN is associated with consumption of dioxygen in a ratio of 1.9 ± 0.2 Fe(II):O2 (
). Furthermore, addition of catalase to the assay solution resulted in the stoichiometric regeneration of O2, indicating production of H2O2 by the ferroxidase reaction as seen with ferritins (
Chen-Barrett Y.
Arosio P.
Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins.
) but not bacterioferritins (
) or Dps ferritins (
Chiancone E.
Stefanini S.
Ilari A.
Iron oxidation and hydrolysis reactions of a novel ferritin from Listeria innocua.
). In contrast, the oxidation stoichiometry of EcFtnA is 3–4 Fe(II) ions per O2, which is suggested to be a consequence of the binding of three iron atoms (one at site C) and a possible fourth iron at an unknown metal site, leading to reduction of O2 to water rather than hydrogen peroxide (
). The ferroxidase reaction in EcFtnA is similar to that observed in human H chain ferritin, although it is more complex due to the third iron in site C (
). Site C, however, is not essential for ferroxidase activity in EcFtnA as site C variants showed only a slight decrease in the overall oxidation rate but the expected stoichiometry of two ferrous iron per dioxygen (
). In contrast, although a third iron site is present in PmFTN, a 2:1 Fe:O2 stoichiometry is retained. The kinetic data reported here support the conclusion that only two Fe(II) ions are initially oxidized per subunit. A key structural difference is that site C in PmFTN is only 3.5–3.7 Å from site B, whereas site C in EcFtnA is 7–8 Å from the A/B pair. Third iron binding sites were observed in EcBFR as well as human mitochondrial ferritin (
Crow A.
Lawson T.L.
Lewin A.
Structural basis for iron mineralization by bacterioferritin.
Langlois d'Estaintot B.
Santambrogio P.
Granier T.
Gallois B.
Chevalier J.M.
Précigoux G.
Levi S.
Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala.
). However, these iron sites were observed to be at the core surface and are more likely involved in the nucleation/mineralization process rather than in ferroxidase center-catalyzed iron oxidation (
Wong S.G.
Tom-Yew S.A.
Mauk A.G.
Structural and mechanistic studies of a stabilized subunit dimer variant of Escherichia coli bacterioferritin identify residues required for core formation.
Only ferroxidase site A is occupied by ferrous iron in the anaerobic crystal; however, stopped-flow data show saturation of the rapid phase 2 after binding of 2 ferrous iron eq per monomer of PmFTN. Together these results point to stepwise binding of the ferrous iron and dioxygen to the ferroxidase site. A model can be proposed in which one ferrous iron binds to site A followed by the binding of the oxidant. Only when the latter is bound can the second ferrous iron bind to site B. Thus, at the moderate iron concentrations (2 mm) used for soaking experiments, a second Fe(II) ion is not observed at the center in the absence of the oxidant (dioxygen). We note that a similar model was proposed for the two Dps proteins from Bacillus anthracis (
Schwartz J.K.
Liu X.S.
Tosha T.
Diebold A.
Solomon E.I.
CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers.
). These are 12-mer (mini)ferritins that contain intersubunit dinuclear ferroxidase centers that are distinct from those of the 24-mer (maxi)ferritins but nevertheless share some common features. For PmFTN, such a model accounts for the observed rate dependence on Fe(II) because once the second Fe(II) binds oxidation can proceed immediately. Thus, ferrous iron binding to site B of the ferroxidase site is proposed to be the rate-determining step.
Evolution of a rapid dioxygen-driven reaction is of value to an organism living in an iron -limited environment. Under low oxygen conditions, a PmFTN 24-mer would sequester at most 24 ferrous ions, making more iron available for metabolic processes. In the presence of dioxygen and an environmental iron pulse, pre-existing PmFTN would rapidly store newly accumulated iron, preventing the generation of reactive oxygen species and providing a buffer for induction of PmFTN expression.
Zn(II) is an inhibitor of EcFtnA and is proposed to compete with ferrous iron for the dinuclear center and consequently inhibit oxidation at these sites (
The use of zinc(II) to probe iron binding and oxidation by the ferritin (EcFtnA) of Escherichia coli.
J. Biol. Inorg. Chem. 1998; 3: 682-688
). In the crystal structure, Zn(II) does bind to sites A and B but is not observed in site C (
). Nonetheless, from the Fe:O2 stoichiometry of the EcFtnA reaction, all three sites were proposed to bind Fe(II) during catalysis. Crystal structures of Zn(II) complexes of EcBFR and human mitochondrial ferritin have Zn(II) bound at sites A and B of the ferroxidase center, consistent with a proposed model of Fe(II) binding (
Willies S.C.
Isupov M.N.
Garman E.F.
Littlechild J.A.
The binding of haem and zinc in the 1.9 Å x-ray structure of Escherichia coli bacterioferritin.
). However, anaerobic Fe(II) complexes for these systems are not available in the literature. We have directly compared Zn(II) and Fe(II) binding in PmFTN (Fig. 2). Zn(II) bound to sites A, B, and C in contrast to the two sites observed in EcFtnA. Furthermore, overall Zn(II) occupancy of the three metal sites resembles iron bound in the presence of dioxygen in PmFTN rather than mimicking Fe(II) binding. Thus, the use of Zn(II) and likely other metal ions as analogs of Fe(II)/Fe(III) may not identify the correct binding sites in other ferritins.
Two models have been proposed for the mechanism of ferroxidation by ferritins and bacterioferritins. In one model, the ferroxidase site functions as a substrate site as seen in human H ferritin (
Hirzmann J.
Mechanism of catalysis of Fe(II) oxidation by ferritin H chains.
), EcFtnA (
Bauminger E.R.
Hechel D.
Nowik I.
Mossbauer spectroscopic investigation of structure-function relations in ferritins.
Biochim. Biophys. Acta. 1991; 1118: 48-58
), and frog M ferritin (
Hwang J.
Krebs C.
Huynh B.H.
Edmondson D.E.
Penner-Hahn J.E.
A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay?.
). Ferrous iron binds to the ferroxidase site, and after oxidation, ferric iron rapidly migrates to the mineral core. In a second model, first described for EcBFR (
) and P. furiosus ferritin (
The dinuclear iron-oxo ferroxidase center of Pyrococcus furiosus ferritin is a stable prosthetic group with unexpectedly high reduction potentials.
FEBS Lett. 2005; 579: 4729-4732
), the ferroxidase site is a stable di-iron site that functions as a cofactor after the binding of 2 eq of ferrous iron per subunit. Additional ferrous ions are then added directly to the mineral core, and the ferroxidase site functions solely in oxygen or peroxide reduction. Recently, Honarmand et al. (
) proposed a unifying mechanism in which the Fe(III) product at the ferroxidase site remains bound to the ferroxidase site but is rapidly displaced by incoming Fe(II). A prediction of this revised model is the observation of a fully Fe(III)-loaded ferroxidase site in crystals after prolonged soaking in Fe(II). The ferroxidase site of PmFTN was not fully occupied after soaking aerobic crystals in ferrous iron for 45 min, suggesting that iron movement occurred at the ferroxidase site during iron loading with 2 mm ferrous iron over an extended time period. Small absorbance changes immediately following oxidation of Fe(II) at the ferroxidase center are consistent with this conclusion. Nevertheless, for PmFTN, the rapid oxidation of ferrous iron was not regenerated upon up to 20 h incubation, indicating that iron remains present at least in part at the ferroxidase site. Thus, if Fe(III) is displaced, the subsequent iron oxidation is much slower than the initial oxidation. Thus, the mechanism of mineralization in PmFTN appears to be more complex with partial iron migration to the core.
Our data indicate that site A of the ferroxidase center has a higher affinity than site B for Fe(II) under anaerobic conditions. The two distinct kinetic phases observed after the second addition of 48 Fe(II) ions may also be related to slow iron migration to the core likely involving the third iron binding site (site C) and perhaps other sites along a path to the cavity. In frog ferritin, the transit of Fe(III) from the ferroxidase center to the cavity has been shown to occur via a pathway through the subunit toward a 4-fold channel (
Felli I.C.
NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin.
Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 545-550
). Site C may function to direct Fe(III) along a different path to the mineral core. In EcBFR, the two ferroxidase center sites were fully occupied after 2.5 min of soaking, and occupancy was not affected following oxidation, suggesting that a distinct mechanism is in operation (
Portions of this work were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the United States Department of Energy Office of Science by Stanford University. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the United States Department of Energy Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393), and the National Center for Research Resources (Grant P41RR001209).
Received in revised form: April 2, 2013
Received: February 5, 2013
The atomic coordinates and structure factors (codes 4ITW, 4IXK, 4ITT, 4IWJ, 4ISP, 4IWK, and 4ISM) have been deposited in the Protein Data Bank (http://wwpdb.org/).


text:
Q: Firebase, listening for a read Is there any way of listening to a read on a reference?
E.g
user1 adds a readListener to ref/books/book1

user2 observes/reads ref/books/book1 and gets its value

user1's readListener is triggered

I haven't seen anything like it in Firebase yet, and I guess a workaround would be to have user2 set some ref/books/book1/read value, but I'm interested to hear if anyone has any other ideas.

A: Firebase is a fairly simple JSON database. There is nothing built-in to fire an event when another user has received specific data.
But as you indeed said, you can build something like that yourself. Most chat applications that need read-confirmation either do this per message or write a "last message ID that I received" marker.

redpajama_set_name: RedPajamaStackExchange


Leap Creek drains a major section of the East slope of the Pine Valley Mountains and empties into Ash Creek to the East of and near exit 33 on Interstate I15.
The Spanish Trail later called the Mormon Trail was an early 19th century north-south route from Salt Lake Utah to Nevada and thence to California.
One north-south route was down the western side of the Pine Valley Mountains through Mountain Meadow, an ideal stopover location with water and grass for cattle. On September 11, 1857 a wagon train of settlers travelling to California was attacked by members of the Mormon Militia together with some Paiute Native Americans. They killed all the adults and older children (totaling about 120 men, women, and children). Seventeen children, all younger than seven, were spared.
Subsequently a north-south route was established down the eastern side of the Pine Valley Mountains. This route was built to service the small communities east of the Pine Valley Mountains but also to avoid the location where the massacre had taken place.
However, this route was blocked by a ridge of volcanic rock which forms the southern edge of the Great Basin. This was the Black Ridge south of Kanarraville.
The major obstacle across the Black Ridge was a deep gorge which required wagons to be disassembled and lowered by ropes. When he first approached this deep gorge, Washington County's first road supervisor, Peter Shirtz, remarked that you could almost leap across the gorge. Thus the location became known as Peters Leap and the waterway through the gorge as Leap Creek.
Later, in 1863, townspeople built the Black Ridge Dugway, near present day I15, through the Black Ridge, permitting wagons to traverse the gorge in one piece. After this, the old route down Peters Leap was abandoned.
Peters Leap and the Leap Creek gorge can be accessed by a rough ATV trail from exit 36 on I15 to the top of the gorge or by travelling up the gorge from an access point at exit 33 on I15.
There are numerous pictographs high on the cliffs in the vicinity of Peters Leap. Below the ridge is a large rock island that reportedly was used by the Paiute Indians as a place where they sacrificed young maidens. Also there is a cave below the ridge where one of the ingredients required to make black powder was mined.
There are two routes to Peters Leap: an overland ATV trail, and a hike up the Leap Creek gorge.
For the overland ATV trail leave Interstate I15 at exit 36 and take old Highway 91 west 1/4 mile to Escalante's Path. Turn left and after 1/2 mile turn left onto 2600E. A short distance along this road the dirt trail to Peters Leap begins. Travel 0.8 miles to an old horse corral. Then take the road west 0.5 miles to the power line service road and follow it south for 1.9 miles until near the gorge. Then take the dirt road that veers to the right 0.2 miles to Peters Leap. This dirt road to Peters Leap is 3.5 miles and is very rough and not passible in wet weather.
To travel up the Leap Creek wash, leave I15 at exit 33 and cross over I15 to the west side and park in the small parking space provided. Hike directly west to the power line, a distance of about 150 feet, to connect to the power line service road which runs south into the Leap Creek Wash.
Here are some pictures from the first part of the trail up the wash.
Here are some pictures of the ATV road to the top.
Here are some pictures from the top of Peters Leap.


text:
Civil rights trail sought in Tuscaloosa
Task force wants to mark area's role
Angel Coker Staff Writer
A group of Tuscaloosa citizens plan to develop a civil rights task force to document and discover Tuscaloosa's history during the civil rights movement and create a trail that includes relevant sites.
"The concept of this is to form a working hub that will collect, preserve and archive" the history of Tuscaloosa's civil rights in order to create a trail, said Scott Bridges, co-chairman of the task force.
Objectives of the task force include documenting existing and new inventory of historic materials, continue collection of oral histories, honor those who participated in civil rights events, make the public aware of these events and present the assembled materials through public programming like lectures and exhibits.
Multiple civil rights events occurred in Tuscaloosa, but Tuscaloosa hasn't truly been recognized along the Alabama Civil Rights Trail.
"If you were to take an official tour (of civil rights historical places in Alabama), there would be no tour for you to take in Tuscaloosa," Bridges said. "There's not a brochure. There's not an app. There's not a tour guide. There's no company that can offer that to you."
"We want to create one in Tuscaloosa for tourism," said city council and task force member Harrison Taylor, who partook in the civil rights events, called Bloody Tuesday.
Bloody Tuesday occurred in 1964 when peaceful black protestors, including Taylor, set out to march from the First African Baptist Church to the Tuscaloosa County Courthouse in an effort to abolish the whites-only drinking fountains.
Those marchers were beaten and tear gassed by law enforcement. Thirty-three black men, women and children were sent to the hospital and another 94 were arrested.
The First African Baptist Church would highlighted on Tuscaloosa's trail because of that event. Foster Auditorium, where Alabama Gov. George Wallace stood in the door to prevent the integration of the University of Alabama by two black students in 1963, would also be on the map.
Other locations could include Linton's Barber Shop, where many of Bloody Tuesday's protestors retreated and the owner has a miniature civil rights museum.
Taylor said another element of the task force may be to create a Tuscaloosa civil rights museum that would also be on the map.
These spots would be recognized by markers and be included on a brochure-type map, he said.
But tourism isn't the only goal, Taylor said. He said he hopes the trail will be a source of education.
Bridges said Tuscaloosa needs to recognize the civil rights events and understand what happened and see how those events affected the future.
"When we connect with history, we connect with memories. We allow ourselves to gain perspective," Bridges said. "If we're not honest about our past, then our solutions to present problems will be skewed."
Reach Angel Coker at angel.coker@tuscaloosanews.com or 205-722-0230.

redpajama_set_name: RedPajamaCommonCrawl


text:
Posted inArchive, News
Brookfield Relay for Life closes in on $1 million mark
Event changes from overnight to daytime this year
by BOB UPHUES July 2, 2012 February 8, 2021
While a 12th anniversary is not normally something to get excited about, that milestone may prove to be monumental for the Brookfield Relay for Life, which is scheduled for Saturday, July 14 at Jaycee-Ehlert Park.
That's because this year the event could reach the $1 million mark in donations since it began back in 2001. As of last week, the Brookfield Relay for Life was just $25,000 short of its goal.
"It's a very achievable goal for this community," said Mary Marshall, who works in the American Cancer Society's Riverside office as an income development representative.
The Relay for Life is a fundraising program for the American Cancer Society. Brookfield is one of some 5,000 communities that host the event annually nationwide. Marshall said she expects about 200 to 250 people to participate in the Brookfield event.
For the first time this year, the Brookfield Relay for Life will be a primarily daytime event. Previously, the local event was an overnight event that started at dusk and continued through to the morning hours.
"We're just looking to get more families involved," said Marshall. "We're hoping people will come throughout the day and participate in the event."
On July 14, the event will kick off officially at 11 a.m. with a survivors' lap and end at 11 p.m., following a luminaria ceremony, where the relay track is lined with illuminated bags dedicated to those fighting cancer or who have succumbed to it.
While some 20 teams comprising about 120 people have been raising funds in the months leading up to the event, the American Cancer Society and the teams themselves will be collecting money during the event.
The event came to Brookfield in 2001, when the American Cancer Society approached then-Brookfield Recreation Department Director Cathy Colgrass Edwards about starting a Brookfield-Riverside event.
Edwards and Michael Garvey, now a village trustee and village president, respectively, were the first co-chairs of the event. That first year, the event raised more than $100,000, helped in part by a $10,000 donation from former Illinois Attorney General Jim Ryan, who is a cancer survivor and whose son was a friend of Garvey's. For Garvey, the event has taken on personal meaning. In 2006, he was diagnosed with cancer and walked in the event subsequently as a cancer survivor.
"There's a lot of focus on the money that's raised, but I have always received a lot of comfort from the event," Garvey said. "It's a great comfort to people who are battling cancer and who have overcome it."
For Edwards, the highlight of the event each year is the luminaria ceremony, which this year will start at 10 p.m. Typically, participants walk the track in silence and the names of those who have battled the disease are read aloud during the ceremony.
"The luminaria announcements are just so touching," Edwards said. "You spend time listening to the names and realizing how many people have been affected by cancer. There's no one that's been untouched by it."


Applications are invited through ONLINE for appointment on vacant posts of Commercial Assistant-I and Commercial Assistant-II in Jaipur Vidyut Vitran Nigam Limited/Ajmer Vidyut Vitran Nigam Limited/Jodhpur Vidyut Vitran Nigam Limited.
Educational Qualifications: Graduation with minimum 60% marks. 5% relaxation in marks shall be allowed to BC/SBC candidates and 10% to SC/ST/PH candidates.
Application Fee: to be deposited in any branch of State Bank of Bikaner and Jaipur, through prescribed challan.
0Responses to "Commercial Assistant-I & II in Vidyut Vitran Nigam Limited Companies of Rajasthan"


text:
mimi in la boheme- peach state opera
Rachel Eve will perform the role of Mimi in Puccini's La Boheme with Peach State Opera. 3pm, Ritz Theatre, Brunswick, Georgia.
mother, amahl and the night visitors, shakespeare opera theatre
Mother, Amahl and the night visitors, shakespeare opere theatre
Rachel Eve will sing the role of the Mother in Menotti's Christmas classic, Amahl and the Night Visitors, with Shakespeare Opera Theatre. Grace the Plains, Virginia, 5pm.
Rachel Eve will sing the role of the Mother in Menotti's Christmas classic, Amahl and the Night Visitors, with Shakespeare Opera Theatre. Grace the Plains, Virginia, 7:30pm.
Countess (cover)-le nozze di figaro-wilmington concert opera
Rachel Eve will be covering the role of the Countess in Wilmington Concert Opera's Le Nozze di Figaro. 2pm, Cathedral of St. Peter, Wilmington, DE.
Countess (cover)-Le nozze di figaro- wilmington concert opera
Rachel Eve is covering the role of the Countess in Wilmington Concert Opera's Le Nozze di Figaro. 7:30pm, Trinity Episcopal Church, Swarthmore, PA.
Cio-cio-san in madama butterfly with capitol city opera
Rachel Eve will perform the role of Cio-Cio-San in Puccini's Madama Butterfly with Capitol City Opera. 7pm, Petite Violette.
cio-cio-san in madama butterfly- capitol city opera
Rachel Eve will perform the role of Cio-Cio-san in Puccini's Madama Butterfly with Capitol City Opera in Atlanta. 7pm, Petite Violette.
soprano soloist-faure requiem
Rachel Eve will be the soprano soloist in Faure's Requiem with Athens First Presbyterian Church, 3pm.
Rachel Eve will perform the role of Mimi in Puccini's La Boheme with Peach State Opera. 3pm, Colquitt County Arts Center, Moultrie, Georgia.
Rachel Eve will perform the role of Mimi in Puccini's La Boheme with Peach State Opera. 3pm, Clayton County Performing Arts Center, Jonesboro, Georgia.
mimi in la boheme- capitol city opera
Rachel Eve will perform the role of Mimi in La Boheme with Capitol City Opera. 7pm, Petite Violette.
mimi in la boheme with capitol city opera
Rachel Eve will perform the role of Mimi in Puccini's La Boheme with Capitol City Opera at Petite Violette in Atlanta, 7pm.
Rachel Eve will perform the role of Mimi in Puccini's La Boheme with Peach State Opera. 3pm, Cole Auditorium, Georgia State Perimeter College.
faculty recital- stafford opera troupe
Rachel Eve will perform opera scenes with fellow faculty members of the Stafford Opera Troupe in Stafford, Virginia. Pieces to be performed include "Mira, o Norma" from Bellini's Norma with mezzo Rebecca Sacks, and "E allor perche" from Leoncavallo's Pagliacci with baritone JP Gorski. The faculty members will be accompanied by pianist Oleg Bellini. 7pm.
in concert, carnegie hall- with pianist greg hankins
Rachel Eve will perform with Greg Hankins at Weill Hall, Carnegie Hall. She will perform alongside the other winners of the 2018 Concert Artists International Virtuoso Competition. Rachel Eve will sing "Absence" by Berlioz. 1pm, Weill Hall, NYC.
fiordiligi in cosi fan tutte- capitol city opera
Rachel Eve will perform the role of Fiordiligi in Mozart's Cosi fan Tutte in Atlanta with Capitol City Opera. 7pm, Petite Violette.
Rachel Eve will perform the role of Fiordiligi in Mozart's Cosi fan Tutte with Capitol City Opera in Atlanta. 7pm, Petite Violette.
Mrs. Anderssen in a little night music- Natchez festival of music
Rachel Eve Holmes performs as Mrs. Anderssen in Sondheim's A Little Night Music.
Natchez Festival of Music
Zweite Dame in Die ZauberflÖte - Arapahoe Philharmonic
Rachel Eve Holmes performs as Zweite Dame (Second Lady) in Mozart's Die Zauberflöte (The Magic Flute) with the Arapahoe Philharmonic in Denver, CO.
April 21, 7:30pm
Soprano solOist in trotta's seven last words
Rachel Eve sings the Soprano Soloist in Michael John Trotta's Seven Last Words with First Baptist Church of Athens choir and orchestra.
March 25, 11:00am
First Baptist Church of Athens
In Recital with pianist Catherine Giel
Warner Robins Community Concert Association
Recital with pianist Catherine Giel
Sunday, March 18, 3pm


ClickFunnels Overview – What you NEED to know!
These tools permit even individuals that have no experience online to be able to create a site and also make it look really good in order to produce a flourishing company that's online. One of those tools is Clickfunnels There is an alternative to Clickfunnels yet usually, this is one of the most popular. This device is among the most effective on the Internet as well as it permits you to actually do a great deal of terrific points with your website.
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The benefit to this item of software application is it comes with a straightforward interface. You can develop a full-page that is click and also go down and you will not have to do anything else in order to build it. There is a great visual interface for you to experiment with and that is all you will certainly ever require. You simply begin by clicking as well as dragging to build your page as if you are putting things together like a problem piece. For somebody that has no technical knowledge, this is a very fundamental part of being able to construct an effective business and build a website that functions well.
If you are searching for a piece of software that will help you grow your business, then you're mosting likely to enjoy Clickfunnels Though if you do not enjoy click for recognizes you need to try to find a Clickfunnels choice.
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Make certain that you are locating a great alternative if you do not let this piece of software. There are many on the marketplace and also you will have the ability to find them quickly.


text:
Q: Define a bijection between $Z^{X \cup Y}$ and $Z^X \times Z^Y$, where $X, Y \in \mathcal{P}(\Omega) $ and $X \cap Y = \emptyset$
Define a bijection between $Z^{X \cup Y}$ and $Z^X \times Z^Y$, where $X, Y \in \mathcal{P}(\Omega) $ and $X \cap Y = \emptyset$

I was given the following task to prove.
I know that we need a map $h : Z^{X \cup Y} \ \to Z^X \times Z^Y$
Firstly, we have to define a function $h(f)$ and the show that is both injective and surjective.
However, I can't define it.
I know that LHS should be $h(f)(x)(y)$. Is it correct ? What should be RHS?

A: Hint: $h(f)=\left(\left.f\right\rvert_X,\left.f\right\rvert_Y\right)$ seems a more reasonable candidate.


Chanel or YSL? What is a girl to do?
I'm in the market for some espadrilles. Does anyone own Chanel or YSL espadrilles? Which do you like most if you own both?
I personally live in my Chanel espadrilles during the spring and summer. It molds to my feet and I can walk hours in them with no problem what so ever. I tried on the YSL espadrilles and didn't like how they felt on my feet. The Chanel's are definitely more comfy (for me at least).
germyom and xpgizzl3 like this.
I tried them on today...fell in love...bought them!
Thank you for your honesty. Lol A year to break shoes in is insane! I bought the Chanels instead.


Auri Vigil, In this week's interview you will learn about an exciting annual event, ExperienceITnm. It is described by the New Mexico Technology Council as an opportunity to "Geek out on all things technology. Our community will gather the best and the brightest entrepreneurs, technicians, hackers, and tech fans for presentations, talks, meet-ups, and parties; all to highlight the vibrant tech community in our city." Auri Vigil joins us to tell us more about the event.
Auri Vigil is the Marketing and Events Coordinator for the New Mexico Technology Council (NMTC). She brings high energy, a positive attitude, and strong work ethic to the Council team. She's well-versed in social media marketing, event coordination, and public relations. With heavy experience in customer relations, small business communications and events, Auri is dedicated to providing Council members, sponsors, and partners with unique event engagement and marketing opportunities.


It's not often that a band can take over a decade off and come back stronger than ever, but with the April 2012 release of their third album Anhedonia, Australian metal band Teramaze has done just that. With driving instrumentation, intricate rhythm work, and adrenaline-pumping vocals, the group (Dean Wells [guitar/backing vocals], Mathew Dawson [guitar], Dean Kennedy [drums], Tijon Lolis [bass], and Brett Rerekura [vocals]) has brought their brand of progressive metal to the rest of the world via Nightmare Records. In this episode of Progtopia, host Mark Ashby speaks with Wells and Lolis about the new album and accompanying video for the title track, what the reason was for such a long hiatus, live gigs in support of bands like Arch Enemy, and the legacy left by their late drummer Julian Percy. Visit the band's website http://teramaze.com.au or the label's website at www.nightmarerecords.com for more information and merchandise.
"Like" Progtopia on Facebook (http://www.facebook.com/Progtopia) and follow Progtopia on Twitter (@Progtopia) to send a message about the show and to receive news about current and upcoming interviews. Please also visit our partner station PRM Radio at www.progrockandmetal.net to listen to and request songs by Teramaze and hundreds of other bands.


Whether youre an entrylevel, professional, or doling out level candidate, we have an HRapproved Apprentice chef resume best suited for you. Job Resume Writing. example of resume for job, resume example for job apply, example resume for job application, resume format, resume template, resume examples, resume contoh, what is resume Apprentice chef resume examples executive chefe how write for work entry level sample cook image cooks professional.
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View. Resume example pastry chef samples cake decorator templates apprentice examples how to craft school. Sample resume for a sous chef How well does your resume sell your culinary skills? Check out this resume sample to see if it could use a few more ingredients to stand out.
Chef resume sample On this page you will find a bank of professionally designed Chef resume templates. Each one if different and written to the highest standards. apprentice chef resume, Getting your CV and cover letter right is a crucial step in applying for any job.
Have a look at our chef resume example written to industry standards that will This free sample resume for a chef has an accompanying sample chef cover letter Essential requirements listed on an Apprentice Chef resume are cooking skills, an interest in learning new things, stamina, flexibility, teamwork, and time management.
Successful example resumes make display of culinary training at an accredited school. Jun 01, 2017 Do you know what to include in your Chef resume? View hundreds of Chef resume examples to learn the best format, verbs, and fonts to use.
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Summary. Apprentice Chef, Apprentice Chef Resume, Apprentice Resume Samples Use this apprentice chef CV template as the starting point for your own jobwinning CV! Customise the template to showcase your experience, skillset and accomplishments, and highlight your most relevant qualifications for a new apprentice chef job.


Program and impressions from the retreat taking place March 19 - 21, 2018.
Schloss Rauischholzhausen, conference venue of the Justus-Liebig-Universität Gießen, situated near the university town of Marburg was the venue of the 2018 SFB Retreat from March 19 - 21, 2018. The detailed program may be found here.
Tim Keiderling from the University if Illinois at Chicago joint our group of nearly 60 SFB 1078 scientists, PIs, Mercator Fellow, postdocs and PhD students. Vivid discussions of talk and poster sessions continued during the guided city tour through Marburg and wine and beer-tasting at the "Market" cellar.


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Lindsay Lohan To Play Sharon Tate?
by Alyssa Caverley at April 5, 2011 12:03 pm . Comments
Lindsay Lohan has been offered the role of murdered actress Sharon Tate, according to Entertainment Weekly.
Tyler Shields tells the magazine the actress is in talks to star as the actress in his upcoming film Eyes of a Dreamer.
The film will focus on serial killer Charles Manson, who famously murdered Tate in her house while she was pregnant - she was married to director Roman Polanski at the time.
"We've worked together many, many times," Shields tells EW. "She asked me about [Eyes] … And I said, 'Oh you should be Sharon Tate.' And she was like, 'That would be amazing.'"
He goes on to explain why he has Lohan in mind saying, "Sharon Tate was a movie star. So I want [an actress who] you already have a backstory with. People have a backstory with someone like Lindsay. And I think it's very relatable."
Shields hopes to start filming July 15, but that could be a problem for Lohan with her currently legal problems. She could be in jail at that time for her felony grand theft charge - the court date is scheduled for April 22.
Shields says he hopes that's not the case and he will be able to work with Lohan on the film.
Tags: Lindsay Lohan, Movie News, Casting News
Lindsay Lohan has had her ups and downs in both her personal life and career. Recently, she's clean up a lot and has been cast in a few films. We're cheering for you Lindsay!
Scary Movie 5 Adds Lindsay Lohan and Charlie Sheen to Cast
Lindsay Lohan Gets Part in Gotti: Three Generations
Joe Pesci Signed to Gotti, Lindsay Lohan in Talks
Lindsay Lohan Photos


While Wolfgang Palm's PPG Phonem synth has been available in VST and AU formats for a while, its recent introduction to the iOS ecosystem is noteworthy, as the features are essentially identical, but the price is only twenty bucks. As a result, Phonem's extraordinary suite of voice synthesis and wavetable tools is now within range of anyone with a newer iPad, so it's worth taking a closer look at what this remarkable app offers.
For starters, Phonem's collection of presets is phenomenal and demonstrates its range admirably. There's a suitable emphasis on spoken and sung vocals, including a stunning soulful melisma called "hohuahou" that uncannily sounds sampled. There's also a great selection of choirs and more traditional synth pads that demonstrate Phonem's potential as a straight-up synth.
For those who like to roll up their sleeves and get busy with sound design, it's crucial to understand Phonem's overall architecture, because it's unlike any other synth you've used.
At the top level is a sequence of phonemes—different vowel and consonant sounds that the human voice produces for language or singing. You can construct phrases using the raw phonemes and dipthongs as syllables (including English and German options) or use the text-to-speech field to enter a phrase and let Phonem do its best to pronounce it properly. This works fairly well in practice, though there's always that "computer accent," which can be finessed using Phonem's other features.
From there, you can adjust the length and formant characteristics of each element in the timeline individually. This really helps with inflection and intelligibility, though it also requires a fair amount of patience, as the process can be a bit tedious. Adding to the complexity is the fact that each phoneme includes its own discrete resonator filter, which is essentially a specialized 12-band EQ for shaping the formant characteristics of each phoneme. This is an extraordinary level of detail for any synth and some users are going to absolutely love it. As a sound designer, I was in heaven.
With all of these specialized filter options, there's obviously a tone-generator in here, right? Well, it is a Wolfgang Palm product, so the oscillator is naturally an incredibly complex wavetable with up to 64 discretely editable waves. In conjunction with the filters, this provides a breathtaking level of nuance. You can also import WTS and TCS files from Palm's WaveGenerator and WaveMapper apps for oscillator information, if you have those available.
Finally, once your phrase is constructed, you can switch to track editing mode and create a fluid pitch sequence that will be applied to it. This is the secret sauce for creating the previously mentioned melisma preset, but in use, you'll quickly discover that pitch inflection is literally "tone of voice." Want to make your phrase into a question? Have the pitch go up at the end. Want it to sound sad? Give it a downward curve. Or you can stick to the basics and just use the track tools to make a musical, singing sequence.
There is a slew of other customizations available for plosives and consonants, LFOs and envelopes for further sculpting, and reverb and delay effects to round out the package.
In thirty-five years of working with synthesizers, I've never encountered a product quite like Phonem. While it is tailored for voice-oriented sound design, the suite of tools is so detailed and specialized that it offers a treasure trove of previously untapped possibilities. This is a synthesis connoisseur's app!
But whether you are looking for new sonic territory to conquer or simply want some cool synthetic vocals, Phonem is worthy of a place on your iPad.
PROS Massive range of phoneme articulations. Integrated phrase sequencer with detailed pitch options.
CONS So many options, designing your own sounds has a significant learning curve.
The deepest voice synthesizer available.


text:
THE WAGE WAR
THE MISSION, A LIVING WAGE
LIVING WAGE CALCULATOR
WAGE SLAVERY
MAKE A POINT
VIDEOS OF THE WAGE WAR ON AMERICANS
JOIN THE WAGE WAR
The income inequality is just un American !
NATIONWIDE BOYCOTT "- – – – WAL-MART"
'Living Wages', Rarity for U.S. Fast-Food Workers, Served Up in Denmark
SOUTH WEST AIRLINES !
TOP 5 WORST EMPLOYERS HIT LIST !
Even talk show host, Steven Colbert gets it !
A study from the University of California, Berkeley found. American fast-food workers earn an average of $8.90 an hour.
THE REAL DEAL ABOUT LIVING ON TIPS !
WALMART "THE TROJAN HORSE OF KILLING SMALL SUCCESSFUL LOCAL BUSINESS" ! <---- Click to see video
HOW YOU ARE JUST EARNING WHAT'S LEFT BEHIND ! <-- click to see video
Airport workers in Seattle take on living wage debate
IS THERE A PILOT SHORTAGE ? OR ARE THE AIRLINES SHOOTING THEMSELVES IN THE FOOT !
CALIFORNIA GIVES CHINA CONTRACT TO BUILD THE SAN FRANCISCO BAY BRIDGE
wage injustice
November 5, 2014 BJ SABER
Eve Edelheit for The New York Times
Our special thanks to Adam Mark for bringing this article to the attention of the wage war website.
If making a living wage in Denmark for fast food worker's is possible, than whats holding us back?
COPENHAGEN — On a recent afternoon, Hampus Elofsson ended his 40-hour workweek at a Burger King and prepared for a movie and beer with friends. He had paid his rent and all his bills, stashed away some savings, yet still had money for nights out.
That is because he earns the equivalent of $20 an hour — the base wage for fast-food workers throughout Denmark and two and a half times what many fast-food workers earn in the United States.
"You can make a decent living here working in fast food," said Mr. Elofsson, 24. "You don't have to struggle to get by."
With an eye to workers like Mr. Elofsson, some American labor activists and liberal scholars are posing a provocative question: If Danish chains can pay $20 an hour, why can't those in the United States pay the $15 an hour that many fast-food workers have been clamoring for?
"We see from Denmark that it's possible to run a profitable fast-food business while paying workers these kinds of wages," said John Schmitt, an economist at the Center for Economic Policy Research, a liberal think tank in Washington.
Many American economists and business groups say the comparison is deeply flawed because of fundamental differences between Denmark and the United States, including Denmark's high living costs and taxes, a generous social safety net that includes universal health care and a collective bargaining system in which employer associations and unions work together. The fast-food restaurants here are also less profitable than their American counterparts.
"Trying to compare the business and labor practices in Denmark and the U.S. is like comparing apples to autos," said Steve Caldeira, president of the International Franchise Association, a group based in Washington that promotes franchising and has many fast-food companies as members.
"Denmark is a small country" with a far higher cost of living, Mr. Caldeira said. "Unions dominate, and the employment system revolves around that fact."
But as Denmark illustrates, companies have managed to adapt in countries that demand a living wage, and economists like Mr. Schmitt see it as a possible model.
Denmark has no minimum-wage law. But Mr. Elofsson's $20 an hour is the lowest the fast-food industry can pay under an agreement between Denmark's 3F union, the nation's largest, and the Danish employers group Horesta, which includes Burger King, McDonald's, Starbucks and other restaurant and hotel companies.
By contrast, fast-food wages in the United States are so low that half of the nation's fast-food workers rely on some form of public assistance, a study from the University of California, Berkeley found. American fast-food workers earn an average of $8.90 an hour.
As a shift manager at a Burger King near Tampa, Fla., Anthony Moore earns $9 an hour, typically working 35 hours a week and taking home around $300 weekly.
To pay fast food restaurant workers a living wage would entail more than raising prices. We would need a national shift in priorities.
"It's very inadequate," said Mr. Moore, 26, who supervises 10 workers. His rent is $600 a month, and he often falls behind on his lighting and water bills. A single father, he receives $164 a month in food stamps for his daughters, 5 and 2. "Sometimes I ask, 'Do I buy food or do I buy them clothes?' " Mr. Moore said. "If I made $20 an hour, I could actually live, instead of dreaming about living."
Mr. Moore's daughters receive health care through Medicaid, while he is uninsured because he cannot afford Burger King's coverage, he said.
"I skip the doctor," he said, adding that he sometimes goes to work sick because "I can't miss the money."
Burger King declined to discuss wages or benefits, saying those decisions were made by its franchise operators. The company said that its "restaurants have provided an entry point into the work force for millions of Americans," and that the Burger King McLamore Foundation gave some employees emergency financial assistance and college scholarships.
Mr. Schmitt, the economist, acknowledged that it would take some time for the American fast-food industry to adjust to higher wages.
"We would need to phase this in," said Mr. Schmitt, who is co-editor of the book "Low-Wage Work in the Wealthy World." "We've created a low-road economy, and it's going to take us some time to build up the speed to get onto the high road."
In Denmark, fast-food workers are guaranteed benefits their American counterparts could only dream of. Under the industry's collective agreement, there are five weeks' paid vacation, paid maternity and paternity leave and a pension plan. Workers must be paid overtime for working after 6 p.m. and on Sundays.
Unlike most American fast-food workers, the Danes often get their work schedules four weeks in advance, and employees cannot be sent home early without pay just because business slows.
Given the benefits available, Mr. Elofsson, the Burger King employee in Copenhagen, said he hoped to make his career with the company and work his way up to restaurant manager. While the Danish industry does not keep data on worker retention, HMSHost Denmark, which runs the fast-food operations at Copenhagen Airport, estimates that 70 percent of the workers at its Burger King and Starbucks franchises stay for more than a year.
By contrast, an internal study done several years ago by McDonald's found that workers' average tenure at the company in the United States was nearly eight months, although the National Restaurant Association said American fast-food workers averaged 20 months on the job.
Danish law does not require fast-food companies or their franchisees to adhere to the wages required by the agreement with the 3F union. But they do, because employees and unions pledge in exchange not to engage in strikes, demonstrations or boycotts. "What employers get is peace," said Peter Lykke Nielsen, the 3F union's chief negotiator with McDonald's.
McDonald's learned this the hard way. When it came to Denmark in the 1980s, it refused to join the employers association or adopt any collectively bargained agreements. Only after nearly a year of raucous, union-led protests did McDonald's relent.
In interviews, Danish employees of McDonald's, Burger King and Starbucks said that even though Denmark had one of the world's highest costs of living — about 30 percent higher than in the United States — their $20 wage made life affordable.
True, a Big Mac here costs more — $5.60, compared with $4.80 in the United States. But that is a price Danes are willing to pay. "We Danes accept that a burger is expensive, but we also know that working conditions and wages are decent when we eat that burger," said Soren Kaj Andersen, a University of Copenhagen professor who specializes in labor issues.
Measured in Big Macs, McDonald's workers in Denmark earn the equivalent of 3.4 Big Macs an hour, while their American counterparts earn 1.8, according to a study by Orley C. Ashenfelter, a Princeton economics professor, and Stepan Jurajda, an economics professor at Charles University in Prague.
And the Danish restaurants are less profitable. With fast-food wages in the United States so much lower than in Denmark, and the price of Big Macs in the two countries similar, Mr. Ashenfelter said, "It must be that U.S. McDonald's are far more profitable." The higher wages and the higher menu prices help explain why there are 16 McDonald's per million inhabitants in Denmark, but 45 McDonald's per million in the United States, Mr. Jurajda said.
McDonald's declined requests for detailed financial data for its restaurants. But it said in a statement that the countries where it operates "have significantly different cost structures, economic environments and competitive frameworks." The company added that it and its franchise operators "support paying valued employees fair wages aligned with a competitive marketplace."
America's restaurant industry predicts a wave of woe if pay were to jump toward Denmark's levels. An increase to $15 would "limit employment opportunities" by making fast-food restaurants reluctant to hire, said Scott DeFife, an executive vice president at the National Restaurant Association. "More than doubling the starting wage will dramatically increase costs in an industry that exists on very narrow margins."
Denmark's high wages make it hard, though not impossible, to maintain profitability at his restaurants, said Martin Drescher, the general manager of HMSHost Denmark, the airport restaurants operator.
"We have to acknowledge it's more expensive to operate," said Mr. Drescher. "But we can still make money out of it — and McDonald's does, too. Otherwise, it wouldn't be in Denmark."
He noted proudly that a full-time Burger King employee made enough to live on. "The company doesn't get as much profit, but the profit is shared a little differently," he said.
"We don't want there to be a big difference between the richest and poorest, because poor people would just get really poor," Mr. Drescher added. "We don't want people living on the streets. If that happens, we consider that we as a society have failed."
Liz Alderman reported from Copenhagen and Steven Greenhouse from New York. Anna-Katarina Gravgaard contributed reporting from Copenhagen.
Previous PostA study from the University of California, Berkeley found. American fast-food workers earn an average of $8.90 an hour.Next PostSELF -CHECK OUT !
Living wage and income eniquality
SELF [DESTRUCTION] CHECK OUT !
GIVE THE FUTURE AMERICANS A CHANCE TO MOVE UP!
ABOUT OUR MISSION, A LIVING WAGE!
Let the WAGE WARS BEGIN !!! November 9, 2015
Nike, "CAPITALISM" at its WORST!!! September 20, 2015
Featured comment-"Online companies like Amazon" Written By Jon September 16, 2015
A Living wage = 1¢ September 7, 2015
America first or is it corporate America first? September 6, 2015
One cent for a Living wage??? August 23, 2015
Unpaid lunch July 27, 2015
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SELF -CHECK OUT ! November 10, 2014
'Living Wages', Rarity for U.S. Fast-Food Workers, Served Up in Denmark November 5, 2014
A study from the University of California, Berkeley found. American fast-food workers earn an average of $8.90 an hour. November 5, 2014
EXAMPLES OF GREAT AMERICANS ! October 18, 2014
CALIFORNIA GIVES CHINA CONTRACT TO BUILD THE SAN FRANCISCO BAY BRIDGE October 17, 2014
"Walmart & Sams Club" October 11, 2014
Even talk show host, Steven Colbert gets it ! October 10, 2014
Airport employees across the the nation fighting for a living wage. October 1, 2014
TOP 5 WORST EMPLOYERS HIT LIST ! September 25, 2014
SOUTH WEST AIRLINES ! September 25, 2014
BJ SABER on SELF [DESTRUCTION] CHECK OUT !
elyse saber-goldstein on SELF [DESTRUCTION] CHECK OUT !
BJ SABER on IS THERE A PILOT SHORTAGE ? OR ARE THE AIRLINES SHOOTING THEMSELVES IN THE FOOT !
Linda M on Nike, "CAPITALISM" at its WORST!!!
Jon on America first or is it corporate America first?
Mathilde Starkey on One cent for a Living wage???
BJ SABER on CALIFORNIA GIVES CHINA CONTRACT TO BUILD THE SAN FRANCISCO BAY BRIDGE
wendy on CALIFORNIA GIVES CHINA CONTRACT TO BUILD THE SAN FRANCISCO BAY BRIDGE
BJ SABER on Report: Walmart Workers Cost Taxpayers $6.2 Billion In Public Assistance
Joe on Report: Walmart Workers Cost Taxpayers $6.2 Billion In Public Assistance


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Video & Text
Annihilate.me
Kris Tralewski
Anamnesis Arcade 3
Rev. 3 of the arcade cabinet arrived as a plywood cabinet with illuminated buttons and a 22" widescreen monitor.
The choice of the finish wasn't accidental – I wanted it to resonate the textures found across the fine art studios, so it signals its affiliation with the art world rather belonging in the amusement arcade. The widescreen version was needed to accommodate a new, higher resolution 3D version of the "Anamnesis Arcade" game. What differentiated it from the previous build was the spherical woodland world equipped with a faux gravity. A world that has undergone an extensive post-processing to replicate the look of a vintage film camera (lens dirt, aberration, bloom and vignetting).
The narrative was handled by the popup windows triggered by the colliders randomly spread across the wooded spherical world. The pop ups showed a collection of photographs referring to the former site of the POW camp Stalag IIIA and screenshots of the unsuccessful Google Maps searches around Luckenwalde. There's very little left on the ground to verify the history.


Travelling Interstate 35 through central Texas, a large Antebellum-style building is sure to catch your eye in Waco. I had seen it before and made a mental note to investigate if I was ever back in the area. So, on a recent road trip through central Texas, we took time to do just that. The beautiful white building is the Collin Street Bakery and, once inside, your senses of smell and taste will be delighted.
The original, world famous DeLuxe fruitcake in its 100th Anniversary tin.
If you're a fan of fruitcake – which I can admit to being – you've probably heard of the Collin Street Bakery. I remember advertising for the "world famous DeLuxe fruitcake" in Sunday papers around the holidays during my youth. Years later, I received one as a business gift and thoroughly enjoyed it. So I was happy to see the sign outside this big white building proclaiming it as the home of the world famous DeLuxe fruitcake. Coincidence? Of course not; I had ingeniously engineered this portion of the road trip to avail ourselves of a visit.
The bakery was founded in Corsicana, Texas – about 55 miles northeast of Waco (and about the same distance south of Dallas). The Waco location is the first outpost, and the staff is every bit as proud of the company's history and fame as the home office is.
Their story is a long and thoroughly enjoyable one that begins with a young German immigrant named August Wiederman who opened the bakery in 1896 with help from local entrepreneur Tom McElwee. After moving to a larger building, McElwee opened a hotel above the bakery, and it attracted renowned celebrities of the day (who likely performed at the opera house he also operated). In 1914, the Ringling Brothers Circus was in town and ordered dozens of fruitcakes to be sent to friends, family and business associates around the United States and Europe. This was their entry into the global mail order business, where they have been phenomenally successful.
When you're "world famous" people find you. This order came from Norway addressed only to "Fruitcake, Texas"!
Each year, some 1.5 million DeLuxe Fruitcakes are sent around the world to a client list that includes descendants of the Ringling Brothers, as well as Princess Caroline of Monaco, who continues her mother, Princess Grace's tradition of sending them as holiday gifts. It is said that the Ayatollah Khomeini of Iran even ordered a fruitcake, though that order went unfilled. In fact, the company claims to ship everywhere except Iran and Cuba.
All of that history and celebrity is built upon fruitcake and there are more varieties than we imagined, all sold only by mail order or at the bakeries. You won't find them anywhere else so, even if you're not a fruitcake fan, you owe it to yourself to try a bite…or several. But save room for all of the other goodies! There are more than sweets and pastries, but that seems to be what they do best. You can get soup and sandwiches (though they don't seem to be made on site in Waco), several types of breads and a variety of coffee drinks.
Yum! This may have been a house built by fruitcake, but the variety of breads, cookies, pastries and more looked delicious! We caved into temptation and took home some raisin bread…and a lot more!
The staff was very friendly, putting up with our indecision with a smile as we tried to narrow down our choices. We decided on several loaves and a souvenir coffee cup for home, and indulged in coffee, strudel and lemon cake to enjoy on the spot…just to confirm that they were every bit as good as the fruitcake we had a sample of. We sat among fellow travelers and a few studious students from nearby Baylor University – apparently nothing fuels the intellect like cinnamon rolls and free Wi-Fi – and enjoyed the big, sunny dining room for a little while before climbing back in the car.
Our recommendation: With free Wi-Fi and excellent pastries, it's a nice place to spend a little time on your way through Waco. You can relax a bit, grab a bite, and take home a true Texas souvenir that you can literally sink your teeth into.
While it's not the original Collin Street Bakery, the Waco location is the first outpost, housed in this beautiful building.
Inside of Collin Street Bakery in Waco, Texas – A comfortable and convenient place for locals, students and travelers.
In addition to sweet treats, you'll find a variety of tempting breads at Collin Street Bakery, too.
You can take the quaint decor home with you! We loved these bread boxes.


text:
Mocking Desire
Drago Jancar
Title: Mocking Desire
Author: Drago Jancar
Original in: Slovene
Availability: Mocking Desire - US
Mocking Desire - UK
Mocking Desire - Canada
Luzifers Lächeln - Deutschland
Slovene title: Posmehljivo pozelenje
Translated from the Slovene by Michael Biggins
From the Northwestern University Press series, Writings from an Unbound Europe
B : solid novel of modern displacement
Rev. of Contemp. Fiction A Spring/1999 Gordon McAlpine
Slavic and E.Europ. Journal . Fall/2000 Helga Glusic
World Lit. Today A Spring/1999 Radmila J. Gorup
Review Consensus:
Very positive.
"(W)hat keeps the novel fresh is the presence of what amounts to an additional central "character, " whose influence is more profound than any other's; that character is Melancholy itself, which changes its appearance from time to time, but hovers nonetheless about every scene in Gradnik's adventure." - Gordon McAlpine, Review of Contemporary Fiction
"This superb exploration of human experience elucidates both the old world from which the author and his protagonist come and the new one they visit." - Radmila J. Gorup, World Literature Today
Mocking Desire is the melancholy-comic story of Gregor Gradnik, a Slovenian writer who gets a fellowship to teach creative writing in New Orleans (apparently mirroring author Jancar's own experiences). He is from a place in the heart of Europe, but it is hardly known: people believe he is from Slovakia, or even "Pennslovenia" (only in America !)
"I am an observer, he tells himself," when he arrives in this strange new world, "I observe what happens to me here on the other side of the world." He remains apart: even after he has settled in his basic character trait remains the same: "He didn't like getting overly involved."
Some involvement is inevitable. There are friendships, fumblings at intimacy, efforts to try to get a grip on this odd place and these odd lifestyles. From the bizarre American idea of "jogging" (which utterly defeats him) to the wilder side of New Orleans (including the usual Mardi Gras excesses), Gradnik does have a fair number of experiences which are related.
Central also is Gradnik's work for Professor Fred Blaumann, also a writer. Blaumann is, quite literally, a blue man, studying and writing about melancholy (and that, of course, in the city of the blues). Robert Burton's The Anatomy of Melancholy (see our review) is a guiding text throughout the book -- so much so that Jancar includes Burton's own detailed charts of melancholy in the novel. Blaumann's project is like a modern version of Burton's classic study, but it too loses itself in melancholy.
Blaumann does also offer writing advice (including sensible advice regarding the dreaded exclamation mark !), though the absurdity of the creative writing programme (and such programmes in general) doesn't concern Gradnik too much. Thankfully, the novel isn't too narrowly focussed on purely writerly concerns.
"Nobody invents anything anymore" is one of the complaints in the novel. Gradnik is also less concerned with invention than observation as he muddles through in the distance, with reality trumping almost all thoughts of invention. He is a stranger in this strange land, but his approach is decidedly that of the visitor rather than exile, only occasionally engaging the world he wanders.
Reality in Slovenia is distant but also inescapable. It intrudes through news from there, and people left behind. Eventually the book wends its way back there too, via the inevitable New York.
There is a fairly colourful cast of characters around Gradnik, and there are some clever episodes, including the ill-fated attempt by one acquaintance (who is also involved in more sordid matters) to found a School for Creative Laughter.
Mocking Desire is a fairly interesting -- and both amusing and poignant -- account of a contemporary (already post-communist) eastern European approach to modern America and a changing world. Gradnik's literary ennui and aimlessness -- a manifestation of the pervasive melancholy all around him -- comes across well. Still, like Gradnik, the novel is a bit unfocussed, unsure of its ends and aims -- which may be part of the point, but isn't always utterly engaging.
Mocking Desire:
Northwestern University Press publicity page
Wieser Verlag publicity page (Slovene)
Wieser Verlag publicity page (German)
Drago Jancar:
Biography at transcript
Interview at transcript (German)
Other books by Drago Jancar under review:
Robert Burton's classic, The Anatomy of Melancholy
Slovenian author Drago Jancar was born 13 April 1948. He has written numerous novels.