Necrotizing sialometaplasia in an HIV positive cocaine user: a case report.

The aim of this paper was to present a case report of a male patient attending a Semiology and Stomatology Clinic with an erythematous ulcerated lesion on his palate. The patient reported that he was HIV positive as well as being addicted to cocaine. After a biopsy and a histopathological exam, he was diagnosed as having necrotizing sialometaplasia. The lesion diminished spontaneously in thirty days after the exam. Correct diagnosis as well as physical and complementary exams are paramount to avoid any incorrect therapy. As drug addiction and HIV infection have both been associated to necrotizing sialometaplasia, as in the present case, it is difficult to establish if the aetiological factor was drug usage or the HIV infection or even, the combination of these two factors. Although considering the influence of HIV infection on the oral health, we may assume that, at least, it favored the onset of this oral lesion.

Crystallization and preliminary crystallographic studies of FoxE from Rhodobacter ferrooxidans SW2, an Fe(II) oxidoreductase involved in photoferrotrophy.

FoxE is a protein encoded by the foxEYZ operon of Rhodobacter ferrooxidans SW2 that is involved in Fe(II)-based anoxygenic photosynthesis (`photoferrotrophy'). It is thought to reside in the periplasm, where it stimulates light-dependent Fe(II) oxidation. It contains 259 residues, including two haem c-binding motifs. As no three-dimensional model is available and there is no structure with a similar sequence, crystals of FoxE were produced. They diffracted to 2.44 Å resolution using synchrotron radiation at the Fe edge. The phase problem was solved by SAD using SHELXC/D/E and the experimental maps confirmed the presence of two haems per molecule.

Experimental study comparing meshes made of polypropylene, polypropylene + polyglactin and polypropylene + titanium: inflammatory cytokines, histological changes and morphometric analysis of collagen.

PURPOSE: Incisional hernia occurs in approximately 11% of all laparotomies. Changes in collagen have been closely implicated in its pathogenesis. The high recurrence rate (45-54%) after primary suture has stimulated the development of meshes. Currently, meshes are the biomaterials implant group most used in medicine. This study aims to compare the serum and tissue inflammatory responses and collagen deposition caused by meshes made of polypropylene, polypropylene + polyglactin and polypropylene + titanium. METHODS: Thirty Wistar rats were divided into three groups. In group I, a high-density polypropylene mesh was positioned on the abdominal wall. In groups II and III, low-density meshes were used in associations with polyglactin and titanium, respectively. Immediately before the operation and on the first, third and fortieth postoperative days, pro-inflammatory cytokines were assayed. On the 40th postoperative day, the region of the inserted prosthesis was biopsied. The tissue inflammatory reaction was evaluated using a scale for objective scoring. For collagen, picrosirius was used with data reading using the Image Tool computer software. RESULTS: Cytokines: there were no statistically significant differences between the groups. HISTOLOGY: on the 40th postoperative day in group I, there were fewer inflammatory tissue response and greater collagen deposition (P < 0.01). In group II, there were greater inflammatory tissue response and less collagen deposition (P < 0.01). Group III presented intermediate values between groups I and II. CONCLUSIONS: There were no significant differences in cytokine levels between the groups in the present study. In the animals with the polypropylene + polyglactin mesh implant there was the most intense inflammatory process with lower tissue maturation and collagen deposition on the 40th postoperative day. The polypropylene mesh presented a less severe late inflammatory process, with greater tissue maturation and collagen deposition. The polypropylene + titanium mesh presented intermediate values between the others.

Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.

The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P2(1) crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P2(1) has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, beta = 105.2 degrees . Model building and refinement are currently under way.

Crystal structure of low-potential cytochrome c549 from Synechocystis sp. PCC 6803 at 1.21 A resolution.

The crystal structure of low-potential cytochrome c549, an extrinsic component of the photosystem II (PS II) from Synechocystis sp. PCC 6803, was obtained directly from single-wavelength 1.21 A resolution diffraction data. This is the first monodomain bis-histidinyl monoheme cytochrome c to be structurally characterized. The extended N-terminal region of c549 builds up a two-strand antiparallel beta-sheet in a hairpin motif, which extends through two molecules owing to crystal packing. Both peptide termini are involved in crystal contacts, which may explain their protrusion out of the globular fold. The C-terminus is preceded by a 9 A-long hydrophobic finger extending from a positively charged base and could be involved in PSII interactions, as well as a protruding negative patch built by a set of conserved acidic residues among c549 sequences.

Structure of a dioxygen reduction enzyme from Desulfovibrio gigas.

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 A resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-beta-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

Crystal structure of cardosin A, a glycosylated and Arg-Gly-Asp-containing aspartic proteinase from the flowers of Cynara cardunculus L.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manbeta4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two beta-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

Ab initio structure solution of a dimeric cytochrome c3 from Desulfovibrio gigas containing disulfide bridges.

The 1.2 A resolution crystal structure of the 29 kDa di-tetrahaem cytochrome c3 from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c3 from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c3 molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.

Crystallization and preliminary diffraction data analysis of both single and pseudo-merohedrally twinned crystals of rubredoxin oxygen oxidoreductase from Desulfovibrio gigas.

Crystals of rubredoxin oxygen oxidoreductase have been obtained and characterized. They belong to space group P2(1)2(1)2, with unit-cell dimensions a = 88.24 (15), b = 101.25 (7), c = 90.80 (3) A. The homodimer (86 kDa) in the asymmetric unit is related by a non-crystallographic twofold rotation axis parallel to the ab 'diagonal' direction, as shown by the self-rotation maximum in the section with chi = 180 degrees. This pseudo-crystallographic symmetry element was also found to be the twinning axis of pseudo-merohedrally twinned crystals, leading to apparent pseudo-tetragonal P42(1)2 crystal symmetry.

Crystallization and preliminary X-ray crystallographic studies of the plant aspartic proteinase cardosin A.

The plant aspartic proteinase cardosin A was crystallized using vapour diffusion. Crystals belong to the monoclinic space group C2, cell dimensions a = 116.9 (2), b = 87.2 (8), c = 81.3 (1) A, beta = 104.4 (4) degrees, and contain two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Diffraction data were collected at room temperature with radiation from a synchrotron source up to 2.85 A resolution. When the crystals were flash cooled to 110 K in a nitrogen stream the same resolution limit could also be obtained on a rotating-anode source. Recently, synchrotron radiation together with flash cooling led to an improvement of the diffraction data to 1.72 A resolution.

The glycosylation of the aspartic proteinases from barley (Hordeum vulgare L.) and cardoon (Cynara cardunculus L.).

Plant aspartic proteinases characterised at the molecular level contain one or more consensus N-glycosylation sites [Runeberg-Roos, P., Tormäkangas, K. & Ostman, A. (1991) Eur. J. Biochem. 202, 1021-1027; Asakura, T., Watanabe, H., Abe, K. & Arai, S. (1995) Eur. J. Biochem, 232, 77-83; Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J. & Pires, E. (1996) Eur. J. Biochem. 235, 762-768]. We found that the glycosylation sites are occupied for the barley (Hordeum vulgare L.) aspartic proteinase (Asn333) and the cardoon (Cynara cardunculus L.) aspartic proteinase, cardosin A (Asn70 and Asn363). The oligosaccharides from each site were released from peptide pools by enzymatic hydrolysis with peptide-N-glycanase A or by hydrazinolysis and their structures were determined by exoglycosidase sequencing combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry. It was observed that 6% of the oligosaccharides from the first glycosylation site of cardosin A are of the oligomannose type. Modified type glycans with proximal Fuc and without Xyl account for about 82%, 14% and 3% of the total oligosaccharides from the first and the second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively. Oligosaccharides with Xyl but without proximal Fuc were only detected in the latter proteinase (4%). Glycans with proximal Fuc and Xyl account for 6%, 86% and 92% of total oligosaccharides from the first and second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively.

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin.

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.

Structure of the tetraheme cytochrome from Desulfovibrio desulfuricans ATCC 27774: X-ray diffraction and electron paramagnetic resonance studies.

The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazão et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three-dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

Cytochrome c6 from the green alga Monoraphidium braunii. Crystallization and preminary diffraction studies.

Cytochrome c(6), a plastocyanin functionally interchangeable electron carrier between the chlorophyll molecule P700 of photosystem I and cytochrome f from cytochrome b(6)f complex, has been isolated from the green alga Monoraphidium braunii and crystallized by the vapour-diffusion technique in sodium citrate. Crystals belong to space group R3, with cell dimensions a = b = 51.93 (5) and c = 80.5 (1) A (hexagonal axes), with one molecule per asymmetric unit. They diffract beyond 1.9 A under a Cu Kalpha rotating-anode source, with an anomalous signal that allows the positioning of the heme Fe atom in the unit cell.

Crystallization and preliminary X-ray diffraction analysis of tetra-heme cytochrome c3 from sulfate- and nitrate-reducing Desulfovibrio desulfuricans ATCC 27774.

Crystals of the tetra-heme cytochrome c(3) (M(r) = 13 kDa, 107 residues, four heme groups) from sulfate- and nitrate-reducing Desulfovibrio desulfuricans ATCC 27774 have been obtained and crystallographically characterized. They belong to space group P6(1)22 with cell dimensions a = b = 61.84 (4) and c = 109.7 (2) A, and Z = 12. Intensity data were initially collected on a FAST system with a rotating-anode X-ray source leading to a total of 22 592 observations, from which only 4930 were unique, in the resolution range 20.0-2.4 A with an R(merge)(I) of 7.0%. Higher resolution data were measured on a FAST system at station 9.6 of the SRS (Daresbury, England), leading to 19 328 intensities, of which 11 179 were unique, in the resolution range 20.0-1.75 A and an R(merge)(I) of 5.5%. Cross-rotation and translation functions were performed with ALMN and TFSGEN programs from the CCP4 suite. The packing of the molecules in the unit cell was checked with TOM/FRODO. Rigid-body refinement of the model and subsequent refinement using molecular dynamics were performed with X-PLOR, leading to a current R factor of 25.9%, for data up to 2.3 A.

X-ray analysis at 2.0 A resolution of mouse submaxillary renin complexed with a decapeptide inhibitor CH-66, based on the 4-16 fragment of rat angiotensinogen.

The structure of mouse submaxillary renin complexed with a decapeptide inhibitor, CH-66 (Piv-His-Pro-Phe-His-Leu-OH-Leu-Tyr-Tyr-Ser-NH2), where Piv denotes a pivaloyl blocking group, and -OH- denotes a hydroxyethylene (-(S)CHOH-CH2-) transition state isostere as a scissile bond surrogate, has been refined to an agreement factor of 0.18 at 2.0 A resolution. The positions of 10,038 protein atoms and 364 inhibitor atoms (4 independent protein inhibitor complexes), as well as of 613 solvent atoms, have been determined with an estimated root-mean-square (r.m.s.) error of 0.21 A. The r.m.s. deviation from ideality for bond distances is 0.026 A, and for angle distances is 0.0543 A. We have compared the three-dimensional structure of mouse renin with other aspartic proteinases, using rigid-body analysis with respect to shifts involving the domain comprising residues 190 to 302. In terms of the relative orientation of domains, mouse submaxillary renin is closest to human renin with only a 1.7 degrees difference in domain orientation. Porcine pepsin (the molecular replacement model) differs structurally from mouse renin by a 6.9 degrees domain rotation, whereas endothiapepsin, a fungal aspartic proteinase, differs by 18.8 degrees. The triple proline loop (residues 292 to 294), which is structurally opposite the active-site "flap" (residues 72 to 83), gives renin a superficial resemblance to the fold of the retroviral proteinases. The inhibitor is bound in an extended conformation along the active-site cleft, and the hydroxyethylene moiety forms hydrogen bonds with both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals contact with groups in the enzyme and define ten specificity sub-sites. This study shows how renin has compact sub-sites due to the positioning of secondary structure elements, to complementary substitutions and to the residue composition of its loops close to the active site, leading to extreme specificity towards its prohormone substrate, angiotensinogen. We have analysed the micro-environment of each of the buried charged groups in order to predict their ionization states.

Structure analysis of cytochrome c3 from Desulfovibrio vulgaris Hildenborough at 1.9 A resolution.

The three-dimensional X-ray structure of cytochrome c3 from sulfate-reducing bacteria Desulfovibrio vulgaris Hildenborough (DvH) (M(r) 13 kDa, 107 residues, 4 heme groups) has been determined at 1.9 A resolution, by the method of molecular replacement, using the homologous part of the refined structure of cytochrome c3 from D. vulgaris Miyazaki F (DvMF). Crystals of c3 DvH were obtained with space group P61, a = 77.0 A, c = 77.2 A, Z = 12, corresponding to two independent molecules in the asymmetric unit. The structure was refined to an R-factor of 19.6%. The structures of the two molecules are analyzed, compared with each other and also with that of c3 DvMF. The main-chain atoms are, for the three structures, generally within 1.0 A. The intramolecular heme edge to edge distances and interplanar angles indicate two groups of values. Shorter distances are associated with near-normal angles, while longer distances with acute angles. Moreover, two of the four hemes, II and IV, are close to only one other heme, while the remaining two hemes, I and III, have two close neighbors each. The two histidine residues that co-ordinate the heme irons on the fifth and sixth positions are nearly parallel, except in the case of heme II. The only substitution from DvMF which is inside the molecule, A68V, occurs in the vicinity of that same heme. However, the non-paralellism between the two flanking histidine residues was also observed in DvMF. Heme II has a conserved higher exposure to solvent and one of the lowest redox potentials in the fully oxidized forms of the two cytochromes. A comparison between data obtained by spectroscopic techniques, nuclear magnetic resonance and electron paramagnetic resonance, and the structural results presented here, indicates two types of interactions, between hemes I and II and between hemes III and IV.

X-ray crystallographic analysis of inhibition of endothiapepsin by cyclohexyl renin inhibitors.

The crystal structures of endothiapepsin, a fungal aspartic proteinase (EC 3.4.23.6), cocrystallized with two oligopeptide renin inhibitors, PD125967 and PD125754, have been determined at 2.0-A resolution and refined to R-factors of 0.143 and 0.153, respectively. These inhibitors, which are of the hydroxyethylene and statine types, respectively, possess a cyclohexylalanine side chain at P1 and have interesting functionalities at the P3 position which, until now, have not been subjected to crystallographic analysis. PD125967 has a bis(1-naphthylmethyl)acetyl residue at P3, and PD125754 possesses a hydroxyethylene analogue of the P3-P2 peptide bond for proteolytic stability. The structures reveal that the S3 pocket accommodates one naphthyl ring with conformational changes of the Asp 77 and Asp 114 side chains, the other naphthyl group residing in the S4 region. The P3-P2 hydroxyethylene analogue of PD125754 forms a hydrogen bond with the NH of Thr 219, thereby making the same interaction with the enzyme as the equivalent peptide groups of all inhibitors studied so far. The absence of side chains at the P2 and P1' positions of this inhibitor allows water molecules to occupy the respective pockets in the complex. The relative potencies of PD125967 and PD125754 for endothiapepsin are consistent with the changes in solvent-accessible area which take place on inhibitor binding.