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PubMed2005

Static versus prospective gated non-breath hold volumetric MDCT imaging of the lungs.

RATIONALE AND OBJECTIVES: The study's aim is to establish lung-imaging methods that provide for the ability to image the lung under dynamic non-breath hold conditions while providing "virtual breath hold" quantifiable volumetric image data sets. Static breath hold images are used as the gold standard for evaluating these virtual breath hold images in both a phantom and sheep. MATERIALS AND METHODS: Axial methods for gating image acquisition to multiple points in the respiratory cycle interleaved with incremental table stepping during multidetector-row computed tomographic (MDCT) scanning were developed. Data sets are generated over multiple breaths, providing volume images representative of multiple points within a respiratory cycle. To determine the reproducibility and accuracy of the methods, six anesthetized sheep were studied by means of MDCT in nongated and airway-pressure (P(awy))-gated modes in which P(awy) was 0, 7, and 15 cm H2O. RESULTS: No significant differences were found between coefficients of variation in air volume measured from repeated static scans (1.74% +/- 1.78%), gated scans: inspiratory (1.2% +/- 0.44%) or expiratory gated (1.39% +/- 0.98%), or between static (1.74% +/- 1.78%) and gated (1.39% +/- 0.98%) scanning at similar P(awy) (P > .1). Measured air volumes were larger from static versus gated scans by 5.85% +/- 3.77% at 7 cm H2O and 4.45% +/- 3.6% at 15 cm H2O of P(awy) (P < .05), consistent with hysteresis. Differences between air volumes at 7 and 15 cm H2O measured from either static or gated scans or that delivered by a super syringe were insignificant (P < .05). Visual accuracy of three-dimensional anatomic geometry was achieved, and landmark certainty was within 1 mm across respiratory cycles. CONCLUSIONS: A method has been shown that provides for accurate gating to respiratory signals during axial scanning. High-resolution volumetric image data sets are achievable while the scanned subject is breathing. Images are quantitatively similar to breath hold images, with differences likely explained by known pressure-volume hysteresis effects.

Animals↗
PubMed2005

Structural evidence for a proton transfer pathway coupled with haem reduction of cytochrome c" from Methylophilus methylotrophus.

The crystal structures of the oxidized and reduced forms of cytochrome c" from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation in the much more complex protein cytochrome c oxidase.

Amino Acid Sequence↗
PubMed1992

Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2000

Evaluation of corneal endothelium and keratic precipitates by specular microscopy in anterior uveitis.

BACKGROUND: The study of corneal endothelium, by specular microscopy, in patients with anterior uveitis has largely been restricted to observations on the endothelial cells. In this prospective study "keratic precipitates" (KP) in different types of uveitis were examined in different stages of the disease process and the endothelial changes occurring in the vicinity of the KP were evaluated in comparison with the endothelium of the uninvolved eye. METHODS: 13 patients with active unilateral uveitis were recruited. The mean age was 42.9 years (range 20-76 years). A Tomey-1100 contact wide field specular (x10) microscope was used to capture endothelial images and KP until the resolution of uveitis. Data regarding type of uveitis, number, size, and nature of KP were recorded. Automated morphometric analysis was done for cell size, cell density and coefficient of variation, and statistical comparisons of cell size and cell density were made (Student's t test) between the endothelium in the vicinity of fresh and resolving KP, fresh KP and normal endothelium, and resolving KP and normal endothelium. RESULTS: On specular microscopy, fresh KP were seen as dense, white glistening deposits occupying 5-10 endothelial cells in diameter and fine KP were widely distributed and were one or two endothelial cells in diameter. The KP in Posner-Schlossman syndrome had a distinct and different morphology. With clinical remission of uveitis, the KP were observed to undergo characteristic morphological changes and old KP demonstrated a large, dark halo surrounding a central white deposit and occasionally a dark shadow or a "lacuna" replaced the site of the original KP. Endothelial blebs were noted as dark shadows or defects in the endothelial mosaic in patients with recurrent uveitis. There was significant statistical difference in the mean cell size and cell density of endothelial cells in the vicinity of fresh KP compared with normal endothelium of the opposite eye. CONCLUSION: This study elucidated the different specular microscopic features of KP in anterior uveitis. Distinct morphological features of large and fine KP were noted. These features underwent dramatic changes on resolution of uveitis. The endothelium was abnormal in the vicinity of KP, which returned to near normal values on resolution of uveitis.

Adult↗
PubMed2005

Synthesis and biodistribution of an 18F-labelled resveratrol derivative for small animal positron emission tomography.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various other plants, and one of the best known 'nutriceuticals'. It shows a multiplicity of beneficial biological effects, particularly, by attenuating atherogenic, inflammatory, and carcinogenic processes. However, despite convincing evidence from experimental and clinical studies, data concerning the role of resveratrol and other members of the large polyphenols family for human health is still a matter of debate. One reason for this is the lack of suitable sensitive and specific methods, which would allow direct assessment of biodistribution, biokinetics, and the metabolic fate of these compounds in vivo. The unique features of positron emission tomography (PET) as a non-invasive in vivo imaging methodology in combination with suitable PET radiotracers have great promise to assess quantitative information on physiological effects of polyphenols in vivo. Herein we describe the radiosynthesis of an (18)F-labelled resveratrol derivative, 3,5-dihydroxy-4'-[(18)F]fluoro-trans-stilbene ([(18)F]-1), using the Horner-Wadsworth-Emmons reaction as a novel radiolabelling technique in PET radiochemistry for subsequent functional imaging of polyphenol metabolism in vivo. In a typical "three-step/one-pot" reaction, (18)F-labelled resveratrol derivative [(18)F]-1 could be synthesized within 120-130 min including HPLC separation at a specific radioactivity of about 90 GBq/mumol. The radiochemical yield was about 9% (decay-corrected) related to [(18)F]fluoride and the radiochemical purity exceeded 97%. First radiopharmacological evaluation included measurement of biodistribution ex vivo and positron emission tomography (PET) studies in vivo after intravenous application of [(18)F]-1 in male Wistar rats using a dedicated small animal PET camera with very high spatial resolution. Concordantly with data on bioavailability and metabolism of native resveratrol from the literature, these investigations revealed an extensive uptake and metabolism in the liver and kidney, respectively, of [(18)F]-1. This study represents the first investigation of polyphenols in vivo by means of PET.

Animals↗
PubMed2001

Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.

Acetylcholine↗
PubMed2003

The design and implementation of a motion correction scheme for neurological PET.

A method is described to monitor the motion of the head during neurological positron emission tomography (PET) acquisitions and to correct the data post acquisition for the recorded motion prior to image reconstruction. The technique uses an optical tracking system, Polaris, to accurately monitor the position of the head during the PET acquisition. The PET data are acquired in list mode where the events are written directly to disk during acquisition. The motion tracking information is aligned to the PET data using a sequence of pseudo-random numbers, which are inserted into the time tags in the list mode event stream through the gating input interface on the tomograph. The position of the head is monitored during the transmission acquisition, and it is assumed that there is minimal head motion during this measurement. Each event, prompt and delayed, in the list mode event stream is corrected for motion and transformed into the transmission space. For a given line of response, normalization, including corrections for detector efficiency, geometry and crystal interference and dead time are applied prior to motion correction and rebinning in the sinogram. A series of phantom experiments were performed to confirm the accuracy of the method: (a) a point source located in three discrete axial positions in the tomograph field of view, 0 mm, 10 mm and 20 mm from a reference point, (b) a multi-line source phantom rotated in both discrete and gradual rotations through +/- 5 degrees and +/- 15 degrees, including a vertical and horizontal movement in the plane. For both phantom experiments images were reconstructed for both the fixed and motion corrected data. Measurements for resolution, full width at half maximum (FWHM) and full width at tenth maximum (FWTM), were calculated from these images and a comparison made between the fixedand motion corrected datasets. From the point source measurements, the FWHM at each axial position was 7.1 mm in the horizontal direction, and increasing from 4.7 mm at the 0 mm position, to 4.8 mm, 20 mm offset, in the vertical direction. The results from the multi-line source phantom with +/- 5 degrees rotations showed a maximum degradation in FWHM, when compared with the stationary phantom, of 0.6 mm, in the horizontal direction, and 0.3 mm in the vertical direction. The corresponding values for the larger rotation, +/- 15 degrees, were 0.7 mm and 1.1 mm, respectively. The performance of the method was confirmed with a Hoffman brain phantom moved continuously, and a clinical acquisition using [11C]raclopride (normal volunteer). A visual comparison of both the motion and non-motion corrected images of the Hoffman brain phantom clearly demonstrated the efficacy of the method. A sample time-activity curve extracted from the clinical study showed irregularities prior to motion correction, which were removed after correction. A method has been developed to accurately monitor the motion of the head during a neurological PET acquisition, and correct for this motion prior to image reconstruction. The method has been demonstrated to be accurate and does not add significantly to either the acquisition or the subsequent data processing.

Algorithms↗
PubMed1984

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.

Apoenzymes↗
PubMed1997

Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria.

On the basis of x-ray diffraction data to a resolution of 2.9 angstroms, atomic models of most protein components of the bovine cytochrome bc1 complex were built, including core 1, core 2, cytochrome b, subunit 6, subunit 7, a carboxyl-terminal fragment of cytochrome c1, and an amino-terminal fragment of the iron-sulfur protein. The positions of the four iron centers within the bc1 complex and the binding sites of the two specific respiratory inhibitors antimycin A and myxothiazol were identified. The membrane-spanning region of each bc1 complex monomer consists of 13 transmembrane helices, eight of which belong to cytochrome b. Closely interacting monomers are arranged as symmetric dimers and form cavities through which the inhibitor binding pockets can be accessed. The proteins core 1 and core 2 are structurally similar to each other and consist of two domains of roughly equal size and identical folding topology.

Animals↗
PubMed1993

Characterization of the post-translational modifications in tubulin from the marginal band of avian erythrocytes.

Tubulin purified from turkey erythrocytes was characterized by partial protein sequence data, high-resolution IEF and by its reaction with antibodies specific for certain post-translational modifications. The tubulin from the marginal band contains a single alpha and beta isotype, i.e. alpha 1 and beta 6. Partial protein sequences and immunoblotting with antibody 6-11B-1 show that erythrocyte alpha 1 tubulin is not acetylated at Lys40. The acidic carboxy-terminal peptides purified by Mono Q chromatography and reverse-phase HPLC were characterized by sequence analysis and mass spectrometry. Although erythrocyte alpha tubulin is almost completely detyrosinated it retains the penultimate glutamic acid residue, which is partially lost in brain tubulin. Thus erythrocyte tubulin is an excellent substrate for extensive in vitro tyrosination by tubulin-tyrosine ligase. Erythrocyte alpha and beta tubulin lack the side-chain polyglutamylation found in all major tubulins from adult brain. Finally we show that about 10% of the beta tubulin is phosphorylated at Ser441. Thus erythrocyte tubulin is an unusual homogeneous preparation. It contains the minimum possible number of tubulin isotypes and the only post-translational modifications detected (detyrosination and phosphorylation) are reversible.

Amino Acid Sequence↗
PubMed1991

Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1.7 A.

The crystal structure of ferredoxin II from Desulfovibrio gigas has been determined using phasing from anomalous scattering data at a resolution of 1.7 A and refined to an R-factor of 0.157. The molecule has an overall chain fold similar to that of the other bacterial ferredoxins of known structure. The molecule contains a single 3Fe-4S cluster with geometry indistinguishable from the 4Fe-4S clusters, and a disulfide bond near the site corresponding to the position of the second cluster of two-cluster ferredoxins. The cluster is bound by cysteine residues 8, 14 and 50. The side-chain of cysteine 11 extends away from the cluster, but could rotate to become the fourth cysteine ligand in the four-iron form of the molecule given a local adjustment of the polypeptide chain. This residue is modified, however, by what appears to be a methanethiol group. There are a total of eight NH . . . S bonds to the inorganic and cysteine sulfur atoms of the Fe-S cluster. There is an additional residue found that is not reported for the chemical sequence: according to the electron density a valine residue should be inserted after residue 55.

Amino Acid Sequence↗
PubMed1995

Crystallization, molecular replacement solution, and refinement of tetrameric beta-amylase from sweet potato.

Sweet potato beta-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm x 0.4 mm x 1.0 mm within 2 weeks, belong to the tetragonal space group P4(2)2(1)2 with unit cell dimensions of a = b = 129.63 A and c = 68.42 A. The asymmetric unit contains 1 subunit of beta-amylase, with a crystal volume per protein mass (VM) of 2.57 A3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric beta-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8-2.3 A (with 2 sigma cut-off) with good stereochemistry. The subunit structure of sweet potato beta-amylase (crystallized in the absence of alpha-cyclodextrin) is very similar to that of soybean beta-amylase (complexed with alpha-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent C alpha atoms of the two beta-amylases is 0.96 A. Each subunit of sweet potato beta-amylase is composed of a large (alpha/beta)8 core domain, a small one made up of three long loops [L3 (residues 91-150), L4 (residues 183-258), and L5 (residues 300-327)], and a long C-terminal loop formed by residues 445-493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (alpha/beta)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (alpha/beta)8 barrel.

Amino Acid Sequence↗
PubMed2003

Novel uncomplexed and complexed structures of plasmepsin II, an aspartic protease from Plasmodium falciparum.

Malaria remains a human disease of global significance and a major cause of high infant mortality in endemic nations. Parasites of the genus Plasmodium cause the disease by degrading human hemoglobin as a source of amino acids for their growth and maturation. Hemoglobin degradation is initiated by aspartic proteases, termed plasmepsins, with a cleavage at the alpha-chain between residues Phe33 and Leu34. Plasmepsin II is one of the four catalytically active plasmepsins that has been identified in the food vacuole of Plasmodium falciparum. Novel crystal structures of uncomplexed plasmepsin II as well as the complex with a potent inhibitor have been refined with data extending to resolution limits of 1.9A and 2.7A, and to R factors of 17% and 18%, respectively. The inhibitor, N-(3-[(2-benzo[1,3]dioxol-5-yl-ethyl)[3-(1-methyl-3-oxo-1,3-dihydro-isoindol-2-yl)-propionyl]-amino]-1-benzyl-2-(hydroxypropyl)-4-benzyloxy-3,5-dimethoxy-benzamide, belongs to a family of potent non-peptidic inhibitors that have large P1' groups. Such inhibitors could not be modeled into the binding cavity of the structure of plasmepsin II in complex with pepstatin A. Our structures reveal that the binding cavities of the new complex and uncomplexed plasmepsin II are considerably more open than that of the pepstatin A complex, allowing for larger heterocyclic groups in the P1', P2' and P2 positions. Both complexed and uncomplexed plasmepsin II crystallized in space group P2, with one monomer in the asymmetric unit. The structures show extensive interlocking of monomers around the crystallographic axis of symmetry, with areas in excess of 2300A(2) buried at the interface, and a loop of one monomer interacting with the binding cavity of the 2-fold related monomer. Electron density for this loop is only fully ordered in the complexed structure.

Amino Acid Sequence↗
PubMed2004

Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding.

The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 A. The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics.

Alcohol Dehydrogenase↗
PubMed1990

High-resolution two-dimensional gel analysis of proteins in wing imaginal discs: a data base of Drosophila.

An improved method of high-resolution two-dimensional gel electrophoresis has been used to study the patterns of protein synthesis in wing imaginal discs of late instar larvae of Drosophila melanogaster. A small number of discs were radiolabeled with a mixture of 14C-labeled amino acids or with [35S]methionine and the pattern of labeled proteins was analyzed. One thousand and twenty-five polypeptides (787 acidic (IEF) and 238 basic (NEPHGE] from wing discs of several wild-type strains have so far been separated and cataloged. All these polypeptides have been numbered and presented in a reference map for further studies. When comparing patterns of label we have found small quantitative differences in rate of synthesis between individuals of the same strain, not due to sexual differences, and very few quantitative and qualitative differences between groups of individuals of different strains.

Amino Acids↗
PubMed1987

Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli.

The protein and solvent structure of the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli yields an R-factor of 0.155 for data to a resolution of 2.6 A. The model has 7353 protein atoms, 945 sites for solvent, and two molecules of CTP. A total of 25 of the 912 residues of the model exist in more than one conformation. The root-mean-square deviation of bond lengths and angles from their ideal values is 0.013 A and 2.1 degrees, respectively. The model reported here reflects a correction in the trace of the regulatory chain. One molecule of CTP binds to each of the two regulatory chains of the asymmetric unit of the crystal. The interactions between the pyrimidine of each CTP molecule and the protein are similar. The 4-amino group of CTP binds to the carbonyl groups of residues 89 (tyrosine) and 12 (isoleucine) of the regulatory chain. The nitrogen of position 3 of the pyrimidine binds to the amide group of residue 12; the 2-keto group binds to lysine 60. The 2'-OH group of the ribose forms hydrogen bonds with lysine 60 and the carbonyl group of residue 9 (valine). The binding of the phosphate groups of CTP to the regulatory chain probably reflects an incomplete association of CTP with the enzyme at pH 5.8. A lattice contact influences the interaction between the triphosphate group of one CTP molecule and the protein. For the other CTP molecule, only lysine 94 binds to the phosphate groups of CTP. Of the two regulatory and two catalytic chains of the asymmetric unit of the crystal, there are only two significant violations of non-crystallographic symmetry. The active site in the vicinity of arginine 54 of one catalytic chain is larger than the active site of its non-crystallographic mate. The "expanded" cavity accommodates four solvent molecules in the vicinity of arginine 54 as opposed to two molecules of water for the "contracted" cavity. Furthermore, arginine 54 in the "expanded" pocket adopts two conformations, either hydrogen-bonding to glutamate 86 or to the phenolic oxygen atom of tyrosine 98; residues 86 and 98 are in a catalytic chain related by 3-fold symmetry to the catalytic chain of arginine 54. In the "contracted" pocket, arginine 54 binds only to glutamate 86.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed2005

Gastro-oesophageal reflux treatment for prolonged non-specific cough in children and adults.

BACKGROUND: Cough is a very common symptom presenting to medical practitioners. Gastroesophageal reflux disease (GORD) is said to be the causative factor in up to 41% of adults with chronic cough. However cough and GORD are common ailments and their co-existence by chance is high. Also cough can induce reflux episodes. Treatment for GORD includes conservative measures (diet manipulation), pharmaceutical therapy (motility or prokinetic agents, H(2) antagonist and proton pump inhibitors (PPI)) and fundoplication. OBJECTIVES: To evaluate the efficacy of GORD treatment on chronic cough in children and adults with GORD and prolonged cough that is not related to an underlying respiratory disease i.e. non-specific chronic cough. SEARCH STRATEGY: The Cochrane Register of Controlled Trials (CENTRAL), the Cochrane Airways Group Specialised Register Collaboration and Cochrane Airways Group, MEDLINE and EMBASE databases, review articles and reference lists of relevant articles were searched. The date of last search was 4th April 2004. SELECTION CRITERIA: All randomised controlled trials on GORD treatment for cough in children and adults without primary lung disease. DATA COLLECTION AND ANALYSIS: Results of searches were reviewed against pre-determined criteria for inclusion. Two independent reviewers selected, extracted and assessed data for inclusion. Authors were contacted for further information. Data was analysed as "intention to treat" as well as "treatment received". Paediatric and adults data were considered separately. Sensitivity analyses were performed. MAIN RESULTS: 11 studies (3 paediatric, 8 adults; 383 participants) were included. None of the paediatric studies could be included in meta-analysis. In adults, analysis on use of H(2) antagonist, motility agents and conservative treatment for GORD were not possible (from lack of data) and there were no controlled studies on fundoplication as an intervention. Five adult studies comparing PPI (2-3 months) to placebo were analysed for various outcomes in the meta-analysis. Enrollment of subjects for two studies were primarily from medical clinics and another three studies were otolaryngeal clinic patients. Using "intention to treat", pooled data from three studies resulted in no significant difference between treatment and placebo in total resolution of cough. Pooled data revealed no significant improvement in cough outcomes (end of trial or change in cough scores). Significant differences were only found in sensitivity analysis. A significant improvement in change of cough scores was found in end of intervention (2-3 months) in those receiving PPI with a standardised mean difference of -0.41 (95%CI -0.75, -0.07) using GIV analysis on cross over trials. Two studies reported improvement in cough after 5 days to 2 weeks of treatment. Significant heterogeneity was found between studies using omeprazole and other PPIs. AUTHORS' CONCLUSIONS: There is insufficient evidence to definitely conclude that GORD treatment with PPI is beneficial for cough associated with GORD in adults. The beneficial effect was only seen in sub-analysis and its effect was small. The optimal duration of such a trial of therapy to evaluate response could not be ascertained in the meta-analysis although two RCTs reported significant change by two weeks of therapy. Clinicians should be cognisant of a period (natural resolution with time) and placebo effect in studies that utilise cough as an outcome measure. Data in children are inconclusive. Future paediatric and adult studies are needed whereby studies should be double blind, randomised controlled, parallel design, using treatments for at least two months, with validated subjective and objective cough outcomes and include ascertainment of time to respond as well as assessment of acid and/or non acid reflux whilst on therapy.

Adult↗
PubMed2006

Lidar characterization of crystalline silica generation and transport from a sand and gravel plant.

Light detection and ranging (Lidar) remote sensing two-dimensional vertical and horizontal scans collected downwind of a sand and gravel plant were used to evaluate the generation and transport of geologic fugitive dust emitted by quarry operations. The lidar data give unsurpassed spatial resolution of the emitted dust, but lack quantitative particulate matter (PM) mass concentration data. Estimates of the airborne PM10 and crystalline silica concentrations were determined using linear relationships between point monitor PM10 and quartz content data with the lidar backscatter signal collected from the point monitor location. Lidar vertical profiles at different distances downwind from the plant were used to quantify the PM10 and quartz horizontal fluxes at 2-m vertical resolution as well as off-site emission factors. Emission factors on the order of 65-110 kg of PM10 (10-30 kg quartz) per daily truck activity or 2-4 kg/t product shipped (0.5-1 kg quartz/t) were quantified for this facility. The lidar results identify numerous elevated plumes at heights >30 m and maximum plume heights of 100 m that cannot be practically sampled by conventional point sampler arrays. The PM10 and quartz mass flux was greatest at 10-25 m height and decreased with distance from the main operation. Measures of facility activity were useful for explaining differences in mass flux and emission rates between days. The study results highlight the capabilities of lidar remote sensing for determining the spatial distribution of fugitive dust emitted by area sources with intermittent and spatially diverse dust generation rates.

Air Pollutants↗