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Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways. The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate. In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor. IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions. Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution. The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073). Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017). A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate. This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain.

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Human primary teeth have been used as indicators of exposure to several heavy metals both in Norway and elsewhere. Local dentists in all 19 counties of Norway collected 2747 primary teeth during 1990-1994. Samples of tooth powder from whole, ground teeth were analyzed for zinc concentration by flame atomic absorption spectrophotometry. The overall geometrical mean was 144.5 micrograms of Zn/g of tooth substance (S.D. = 1.6). The result represents a small increase (5.2%) compared with a similar investigation in the 1970s. However, the mean zinc concentrations in the geographically matching parts of the two materials did not differ significantly. The variation in tooth zinc concentrations between the different counties declined from the 1970s to the 1990s. We found no correlation between the tooth zinc concentration and available environmental data on zinc in drinking-water, discharge of zinc from industrial point sources or population density in the same geographical areas. The zinc concentrations varied significantly with caries status, tooth type and root length. Few samples had a zinc concentration below 90 micrograms/g, indicating that most children consume sufficient zinc. Some very high values could not immediately be explained, but may be caused by contamination from zinc-containing dental restorations.

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Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.

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The soil-dwelling microbe, Pseudomonas sp. strain CBS-3, has attracted recent attention due to its ability to survive on 4-chlorobenzoate as its sole carbon source. The biochemical pathway by which this organism converts 4-chlorobenzoate to 4-hydroxybenzoate consists of three enzymes: 4-chlorobenzoyl-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoyl-CoA thioesterase. Here we describe the three-dimensional structure of the thioesterase determined to 2.0-A resolution. Each subunit of the homotetramer is characterized by a five-stranded anti-parallel beta-sheet and three major alpha-helices. While previous amino acid sequence analyses failed to reveal any similarity between this thioesterase and other known proteins, the results from this study clearly demonstrate that the molecular architecture of 4-hydroxybenzoyl-CoA thioesterase is topologically equivalent to that observed for beta-hydroxydecanoyl thiol ester dehydrase from Escherichia coli. On the basis of the structural similarity between these two enzymes, the active site of the thioesterase has been identified and a catalytic mechanism proposed.

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The structure of rabbit muscle pyruvate kinase crystallized as a complex with Mg2+, K+, and L-phospholactate (L-P-lactate) has been solved and refined to 2.7 A resolution. The crystals, grown from solutions of polyethylene glycol 8000 at pH 7.5, belong to the space group P2(1) and have unit cell parameters a = 144.4 A, b = 112.6 A, c = 171.2 A, and beta = 93.7 degrees. The asymmetric unit contains two tetramers. The crystal structure reveals that the eight subunits within the asymmetric unit adopt several different conformations. These conformations are characterized by differences in the relative positions of protein domains A and B, resulting in different degrees of closure of the active site cleft that occupies the interface between these two domains. The global conformational differences may be described as rotations of the B domain with respect to the (beta/alpha)8-barrel of the A domain. Carbon atoms of the backbone in domain B rotate >20 degrees from the most open to the most closed subunit. The different conformations among subunits within the asymmetric unit are accompanied by 3-3.8 A shifts in the position of Mg2+ and a significant change in the orientation of the phenyl ring of Phe 243. In all of the subunits, Mg2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. In the subunit having the most closed conformation, Mg2+ also coordinates to the carboxylate oxygen, the bridging ester oxygen, and a nonbridging phosphoryl oxygen of L-P-lactate. Mg2+ to L-P-lactate coordination is missing in subunits exhibiting a more open conformation. K+ coordinates to four protein ligands and to a phosphoryl oxygen of the L-P-lactate. The position and liganding of K+ are unaffected by the different conformations of the subunits. The side chain of Arg 72, Mg2+, and K+ provides a locus of positive charge for the phosphate moiety of the analog in the closed subunit.

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UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-galactose and UDP-glucose through the transient reduction of the tightly bound cofactor NAD+. The enzyme is unique among the NAD+-dependent enzymes in that it promotes stereospecific reduction of the cofactor but nonstereospecific hydride return during normal catalysis. In addition to hydride transfer, the reaction mechanism of epimerase involves two key features: the abstraction of a proton from the 4'-hydroxyl group of glucose or galactose by an active site base and the rotation of a 4-ketopyranose intermediate in the active site pocket. To address the second issue of movement within the active site, the X-ray structures of reduced epimerase complexed with UDP-mannose, UDP-4-deoxy-4-fluoro-alpha-D-galactose, or UDP-4-deoxy-4-fluoro-alpha-D-glucose have been determined and refined to 1.65, 1.8, and 1.65 A resolution, respectively. A comparison of these models to that of the previously determined epimerase/NADH/UDP-glucose abortive complex reveals that the active site accommodates the various sugars by simple rearrangements of water molecules rather than by large changes in side chain conformations. In fact, the polypeptide chains for all of the epimerase/NADH/UDP-sugar complexes studied thus far are remarkably similar and can be superimposed with root-mean-square deviations of not greater than 0.24 A. The only significant differences between the various enzyme/UDP-sugar models occur in two of the dihedral angles defining the conformation of the UDP-sugar ligands.

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Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.

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Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

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Lead is one of the most important and widely distributed pollutants in the environment. In the human population children are particularly at risk. Local dentists in all 19 counties in Norway collected 2746 primary teeth from 1990 to 1994. Tooth substance from whole, ground teeth were analyzed for lead concentration by electrothermal atomic absorption spectrophotometry. The geometrical mean for the entire material was 1.27 micrograms lead/g tooth substance (S.D. 1.87). The mean lead levels in each of the counties were significantly lower than those obtained in a corresponding study in the 1970s. Two counties, Oslo and Vest-Agder, had significantly higher lead levels than the majority of the other counties. Tooth lead concentration and atmospheric deposition of lead in the same areas were significantly and positively correlated, as shown by analysis of naturally growing moss. We conclude that lead concentrations in primary teeth from children in Norway have been reduced by approx. 50% from the 1970s to the 1990s. The reduction probably reflects a decrease in the environmental lead burden in Norway.

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Myosin light-chain kinase is responsible for the phosphorylation of myosin in smooth muscle cells. In some tissue types, the C-terminal portion of this large enzyme is expressed as an independent protein and has been given the name telokin. Recently, an antibody directed against telokin was found to interact with a protein derived from the baculovirus Autographa californica nuclear polyhedrosis virus. This protein was biochemically characterized and given the name TLP20 for telokin-like protein of 20 000 molecular weight. The amino-acid sequence of TLP20 was determined on the basis of a cDNA clone and subsequent alignment searches failed to reveal any homology to telokin or to other known proteins. The three-dimensional structure of a proteolytic portion of TLP20 is reported here. Crystals employed in the investigation were grown from ammonium sulfate solutions at pH 6.0 and belonged to the space group P2(1)3 with unit-cell dimensions of a = b = c = 76.3 A and one molecule per asymmetric unit. The structure was determined by multiple isomorphous replacement with three heavy-atom derivatives. Least-squares refinement of the model reduced the crystallographic R factor to 18.1% for all measured X-ray data from 30.0 to 2.2 A. The overall fold of the molecule may be described as a seven-stranded antiparallel beta-barrel flanked on the bottom by two additional beta-strands and on the top by an alpha-helix. Quite surprisingly, the three-dimensional structure of this beta-barrel is not similar to telokin or to any other known protein.

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Here we describe the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3. This enzyme catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. The molecular structure of the enzyme/4-hydroxybenzoyl-CoA complex was solved by the techniques of multiple isomorphous replacement, solvent flattening, and molecular averaging. Least-squares refinement of the protein model reduced the crystallographic R factor to 18.8% for all measured X-ray data from 30 to 1.8 A resolution. The crystallographic investigation of this dehalogenase revealed that the enzyme is a trimer. Each subunit of the trimer folds into two distinct motifs. The larger, N-terminal domain is characterized by 10 strands of beta-pleated sheet that form two distinct layers which lie nearly perpendicular to one another. These layers of beta-sheet are flanked on either side by alpha-helices. The C-terminal domain extends away from the body of the molecule and is composed of three amphiphilic alpha-helices. This smaller domain is primarily involved in trimerization. The two domains of the subunit are linked together by a cation, most likely a calcium ion. The 4-hydroxybenzoyl-CoA molecule adopts a curved conformation within the active site such that the 4-hydroxybenzoyl and the adenosine moieties are buried while the pantothenate and pyrophosphate groups of the coenzyme are more solvent exposed. From the three-dimensional structure it is clear that Asp 145 provides the side-chain carboxylate group that adds to form the Meisenheimer intermediate and His 90 serves as the general base in the subsequent hydrolysis step. Many of the structural principles derived from this investigation may be directly applicable to other related enzymes such as crotonase.

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Rat molars are indicators of exposure concentration and target organ content in chronic mercury vapor exposure. We wished to study the accumulation and persistence of organic and inorganic mercury in rat teeth and the effect of selenium on mercury retention. Male Wistar rats received either inorganic or organic mercury (with or without addition of selenite), selenite only, or no mercury or selenite (controls) in the drinking water for 4 weeks. Group A was killed after exposure. Group B was killed 20 weeks later. The mercury content was measured by cold-vapor atomic absorption spectrophotometry. The mercury content in the molars in group B was 66% and 77% less than in group A after inorganic and organic exposure, respectively. In the incisors the corresponding reductions were 90% and 97%. Selenite had limited effect on mercury retention in group A and none in group B. We suggest that rat molars and, by inference, human deciduous teeth may serve as indicators of organic and inorganic mercury exposure.

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The three-dimensional structure of the fatty-acid-binding protein isolated from the flight muscle of the desert locust Schistocerca gregaria has been solved and refined to a crystallographic R-value of 18.5% for all measured X-ray data from 30.0- to 2.2-A resolution. Crystals employed in the investigation were grown from 2.6 to 2.8 M ammonium sulfate solutions, buffered at pH 7.5 and containing 2-5% 2-methyl-2,4-pentanediol. They belonged to the space group P2(1) with unit cell dimensions of a = 61.6 A, b = 44.8 A, c = 63.9 A, and beta = 113.6 degrees and two molecules per asymmetric unit. The protein fold consists of ten strands of antiparallel beta-pleated sheet that wrap around to form a beta-barrel. In addition, there are two small alpha-helices and six type I, two type II, and two type II' turns. The two molecules pack in the asymmetric unit as a dimer with a local 2-fold rotational axis. The subunit-subunit interface involves amino acid side chains located in the area of the helix-turn-helix motif and the turn between beta-strands E and F. It is this area that has been speculated to form the portal through which fatty acids enter the binding cavity. There are 23 solvent molecules that are conserved between the two independent molecules in the asymmetric unit. Nine of these waters play important structural roles. A three-dimensional comparison between the insect and human muscle fatty-acid-binding proteins shows that their alpha-carbons superimpose with a root-mean-square deviation of 0.77 A for 89 structurally equivalent atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

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Male, adult Wistar rats were exposed to 500 micrograms/m3 mercury vapor 6 h per day, 5 days a week for 4 wk. They were subsequently killed by transcardial perfusion. The molars were extracted, demineralized, and embedded in resin before sectioning. Autometallographic development was performed according to the method of Danscher & Möller-Madsen. Mercury deposits were found in small amounts in several areas of the pulp, but with larger accumulations of grains in relation to odontoblasts. Mercury also could be seen in odontoblastic processes in the dentin and predentin. Our conclusion is therefore that systemic uptake of mercury vapor leads to accumulation of mercury in the odontoblasts and that the mercury may be transported into the dentin tubules in the odontoblastic process.

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Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described. This structure of a molecular motor was determined by single crystal x-ray diffraction. The data provide a structural framework for understanding the molecular basis of motility.

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Five groups of Wistar rats received graded concentrations of mercury vapor from 10 to 100 micrograms Hg/m3 6 hr, 5 days a week, from 4 to 11 weeks of age. One group breathing ambient air served as controls. The mercury levels of the indicators blood, hair, molars, and incisors as well as the target organs kidney cortex, cerebrum, cerebellum, liver, lung, spleen, tongue, and femur were measured by cold-vapor atomic absorption spectrophotometry. The mercury vapor had no negative influence on the weight gain of the animals. The results showed that the kidney cortex had the highest concentration of mercury. The mercury contents of all the indicators and all the target organs, with the exception of femur, were positively and significantly correlated with the exposure concentration. The rat molars had the highest correlation coefficient with the kidney mercury values, but no indicator had a significant correlation with all target organs. Rat molars are to some degree comparable to human deciduous teeth regarding time of mineralization and eruption. Based on the results presented in this study, we tentatively suggest that human deciduous teeth can be useful indicators of chronic mercury exposure not only at the exposure concentration level, but also as indicators of the mercury uptake in organs such as kidney and cerebrum.

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Seventy-nine primary (deciduous) teeth were excavated in 1978 underneath the floor of the stave church in Uvdal, Buskerud County, Norway. The mercury content of 57 teeth was measured by means of cold vapor atomic absorption spectrophotometry. As a comparison, 124 primary teeth from modern Norway were analyzed. A significant statistical difference was found between the two sets of material. In the Uvdal material a correlation was found between the mercury and copper contents. For the modern material a correlation was found between mercury and lead, and between mercury and zinc. The authors maintain that the values found for the Uvdal material represent base-line values for mercury in primary teeth, and probably reflect uptake from natural environmental sources only. Furthermore, these values may be used for reference in studies of other preindustrial, as well as modern, primary teeth. Our findings also indicate a higher level of mercury in modern than in preindustrial primary teeth in Norway.

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The molecular structure of the high-potential iron-sulfur protein (HiPIP) isolated from the phototrophic bacterium, Rhodocyclus tenuis, has been solved and refined to a nominal resolution of 1.5 A with a crystallographic R-factor of 17.3% for all measured X-ray data from 30 A to 1.5 A. It is the smallest of the HiPIP structures studied thus far with 62 amino acid residues. Crystals used in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A and beta = 90.8 degrees and contained two molecules per asymmetric unit. The structure was solved by a combination of multiple isomorphous replacement with two heavy-atom derivatives, anomalous scattering from the iron-sulfur cluster, symmetry averaging and solvent flattening. The folding motif for this HiPIP is characterized by one small alpha-helix, six Type I turns, an approximate Type II turn and one Type I' turn. As in other HiPIPs, the iron-sulfur cluster is co-ordinated by four cysteinyl ligands and exhibits a cubane-like motif. These cysteinyl ligands are all located in Type I turns. The hydrogen bonding around the metal cluster in the R. tenuis protein is similar to the patterns observed in the Chromatium vinosum and Ectothiorhodospira halophila HiPIPs. Several of the amino acid residues invariant in the previously determined C. vinosum and E. halophila structures are not retained in the R. tenuis molecule. There are 13 solvent molecules structurally conserved between the two R. tenuis HiPIP molecules in the asymmetric unit, some of which are important for stabilizing surface loops. Interestingly, while it is assumed that this HiPIP functions as a monomer in solution, the two molecules in the asymmetric unit pack as a dimer and are related to each other by an approximate twofold rotation axis.

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The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.

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