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Biomedical subjects

L Watanabe

Publications and source records attributed to L Watanabe.

At least 19 recordsLinked to original sources
PubMed2003

Initial structural analysis of an alpha4beta4 C-type lectin from the venom of Crotalus durissus terrificus.

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 A, and diffraction data were collected to 2.7 A. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.

Animals↗
PubMed2003

Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida is a key enzyme in the Entner-Doudoroff pathway which catalyses the cleavage of KDPG via a class I Schiff-base mechanism. The crystal structure of this enzyme has been refined to a crystallographic residual R = 17.1% (R(free) = 21.4%). The N-terminal helix caps one side of the torus of the (betaalpha)(8)-barrel and the active site is located on the opposite, carboxylic side of the barrel. The Schiff-base-forming Lys145 is coordinated by a sulfate (or phosphate) ion and two solvent water molecules. The interactions that stabilize the trimer are predominantly hydrophobic, with the exception of the cyclically permuted bonds formed between Glu132 OE1 of one molecule and Thr129 OG1 of a symmetry-equivalent molecule. Except for the N-terminal helix, the structure of KDPG aldolase from P. putida closely resembles the structure of the homologous enzyme from Escherichia coli.

Aldehyde-Lyases↗
PubMed2003

Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid.

Lys49-Phospholipase A2 (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region permits quaternary structural transitions between "open" and "closed" membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78A, (ii) MjTX-II/STE complex at a resolution of 1.8 A and (iii) BthTX-I/DMPC complex at 2.72A. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (iii) and using using a Synchrotron Radiation Source (Laboratório Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).

Animals↗
PubMed2002

Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus.

Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 Da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 A, beta = 111.32 degrees, and diffract to a nominal resolution of 1.61 A. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 A, beta = 125.55 degrees, and diffract to a nominal resolution of 2.78 A. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.

Amino Acid Sequence↗
PubMed2002

Crystallization and preliminary diffraction data of BaP1, a haemorrhagic metalloproteinase from Bothrops asper snake venom.

BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 A. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 A.

Animals↗
PubMed2002

Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca.

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogenous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 A. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 A.

Animals↗
PubMed2002

Transvaginal ultrasonography in predicting placenta previa at delivery: a longitudinal study.

OBJECTIVES: To determine the extent of overlapping of the internal cervical os by the lower placental edge at 11-14 weeks' gestation which best predicts placenta previa at term. PATIENTS AND METHODS: This was a prospective study initially involving 381 singleton pregnancies with a live fetus at 11-14 weeks attending for routine antenatal care. The distance between the lower placental edge and the internal cervical os was longitudinally evaluated by transvaginal ultrasound examination at 11-14 weeks', 20-24 weeks', and 30-34 weeks' gestation. The first 203 cases were selected at random (first phase) and after this period only cases with the lower placental edge reaching and/or overlapping the internal cervical os were followed up (n = 170, second phase). Thus a total of 373 cases were analyzed, 351 of whom were examined in all three trimesters. Multiple regression analysis was used to estimate the probability of predicting placenta previa at term using the overlap of the lower placental edge over the internal cervical os in the first trimester of pregnancy. RESULTS: A change in the relative position of the placenta (placental migration) was observed in all 351 cases examined in the three trimesters of pregnancy. In the general population, represented by the 203 cases (first phase), the incidence of placenta previa at 11-14 weeks' gestation was 42.3% (86/203), at 20-24 weeks' 3.9% (8/203) and at term 1.9% (4/203). A total of 18 cases of placenta previa and 17 cases of marginal placenta were observed at term. It was estimated that when the lower placental edge overlaps the internal cervical os by 23 mm at 11-14 weeks the probability of placenta previa at term is 8% with a sensitivity of 83.3% and specificity of 86.1%. CONCLUSION: The present study establishes the probability of placenta previa at term depending on the relationship of the lower placental edge to the internal cervical os at 11-14 weeks.

Adolescent↗
PubMed2000

Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber.

ADP-glucose pyrophosphorylase is the key regulatory enzyme in the biosynthesis of starch in plants and glycogen in bacteria. The enzyme from potato tuber is comprised of a regulatory subunit and a catalytic subunit and is present as a heterotetramer (alpha(2)beta(2)). The catalytic subunit from potato tuber (50 kDa) was crystallized in four different forms, two of which are suitable for structural studies. A tetragonal crystal form obtained in the presence of the substrate analog Cr-ATP diffracted to 2.2 A and belongs to space group P4(1) (or its enantiomorph), with unit-cell parameters a = b = 110.57, c = 190.14 A. A second crystal form obtained diffracted to 2.8 A and belongs to space group P2, with unit-cell parameters a = 80.06, b = 138.84, c = 92.20 A, beta = 112. 40 degrees. As this protein displays no significant homology to any currently known protein structure, a search for heavy-atom derivatives has been initiated.

Adenosine Triphosphatases↗
PubMed1999

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis.

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

Aspergillus oryzae↗
PubMed1997

Three-dimensional images of the mouse submandibular gland: an HRSEM study.

The three-dimensional characteristics of the intracellular components of acinar cells were revealed using the osmium-DMSO-osmium method. The specimens were macerated in diluted osmium after being fractured in DMSO solution. The stacks of the Golgi apparatus, the rough endoplasmic reticulum and mitochondria are clearly seen. The lamellae of the rough endoplasmic reticulum, located around the nuclei and the Golgi apparatus, are shown in three-dimensional images. At high magnification, the mitochondria clearly reveal their cristae mitochondriales.

Animals↗
PubMed1992

In vitro microleakage at the gingival margin of porcelain and resin veneers.

This in vitro study investigated the effect of dental surface treatments on the seal of porcelain and resin laminate veneers to tooth structure. Nine groups of 20 teeth were prepared for labial veneers--six groups with the apical margin of the veneer in dentin, two groups with the veneer wholly bordered by enamel, and one group with the apical margin coincident with the apical margin of a previously placed glass ionomer restoration. The teeth were treated with a variety of dentin-bonding agents prior to restoration of eight groups with porcelain veneers and one group with Visio-Gem microfilled resin veneers. After placement, the veneers were stored in water for 4 days, then subjected to thermocycling before silver staining and sectioning. Results showed no leakage around the margins of the veneers wholly bordered by enamel. Complete leakage was found around the glass ionomer restorations, and nearly total leakage was found along the resin-dentin interface of the resin veneers. The porcelain veneers that extended onto dentin showed variable but limited leakage for four of five of the dentin-bonding treatments.

Acid Etching, Dental↗
PubMed1992

Comparison of tensile strength of solder joints by infrared and conventional torch technique.

Fabrication of a fixed partial denture may require a soldering step. This study compared soldering by a conventional torch procedure with an infrared soldering technique. Comparisons were made for tensile strength, porosity, and time efficiency between the two methods. No significant difference was found in ultimate tensile strength between the two types of solder joints and the nonsoldered control samples. Random samples photographed with a scanning electron microscope revealed no difference in joint porosity between the two techniques. Torch soldering took consistently less time that infrared soldering.

Dental Alloys↗
PubMed1992

Use of self-curing composite resins to facilitate amalgam repair.

Resin cements, which have been shown to adhere to various metal alloys, were investigated as possible repair adhesives for dental amalgam. Test bars of repaired amalgam, formed by condensing new alloy against previously set alloy with or without the use of "adhesive" resins, were subjected to three-point bending measurements of transverse fracture strength. The results indicated that application of adhesive resin did not improve the breaking strength of the repaired specimens from that of specimens prepared without the use of such resins. The breaking strength of all repaired specimens was approximately 15% of the transverse fracture strength of the original intact amalgam bars. Scanning electron microscopy revealed that there was an intermixing of fresh amalgam and unset interfacial resin, which led to mechanical bonding of these materials, but there was no evidence of adhesion of the resin to the previously set amalgam.

Acrylic Resins↗
PubMed1990

Pulp chamber temperature changes with visible-light-cured composites in vitro.

Due to the low compliance nature of the dental pulp, it is vulnerable to cavity preparation and restoration procedures. The vulnerability may be due to temperature increases within the pulp tissue generated by those procedures. Placement of composites includes the use of visible-light-cure lamps which emit heat and cause a temperature increase in the pulp. This study measured the temperature increase occurring in the pulp chamber with the placement of a two-surface posterior composite using six visible-light-cure lamps. The data indicate that the lamps vary in the amount of temperature increase generated from one to another; that the greater portion of the temperature increase occurred during the placement of the first layer of composite; and that one lamp caused significantly lower temperature increases.

Composite Resins↗
PubMed1990

Evaluation of temperature rise on the outer surface of teeth during root canal obturation techniques.

The heat generated at the apical 2 mm of the outer root surface of extracted human canine teeth during three obturation procedures was measured. The rise in temperature was found to be less when a sealer was used in all obturation techniques. Temperatures as high as 44.02 degrees C were recorded with warm gutta-percha with no sealer, and the lowest temperature rise occurred with a thermoplasticized gutta-percha technique with a sealer. The thickness of the remaining dentin and cementum after cleaning and shaping were found to have no effect in reducing temperature rise. The results of this investigation indicated that temperature rise on the outer surface of the root as a result of heat-generating obturation procedures is negligible and will not have an effect on the supporting attachment apparatus.

Bismuth↗
PubMed1989

An evaluation of sealing ability of calcium hydroxide sealers.

Solid core filling material such as gutta-percha has been used to fill the root canal in conjunction with a sealer to prevent apical leakage. The purpose of this study was to compare apical seal of five different root canal sealers. Sixty roots of maxillary central incisors were cleansed and shaped and randomly assigned to one of six groups of ten roots each. The root canals were obturated with gutta-percha and one of the sealers by the lateral-vertical condensation technique. Groups I, II, III, IV, and V consisted of roots in which the canal was filled with gutta-percha along with either Roth's sealer, AH26, Sealapex, CRCS, or Nogenol sealer, respectively. Group VI was filled with gutta-percha and without sealer to serve as a control. The access opening was filled with amalgam. Each tooth was then placed in a capped vial containing 2 X 2 inch gauze pads saturated with distilled water, and the sealer was allowed to set at 37 degrees C in the humidor for 48 hours. The roots, except for the apical 2 mm, were coated with two layers of nail polish. The roots were stained with india ink, decalcified, dehydrated, and then placed in methylsalicylate to make them transparent. The leakage was measured with a filar micrometer eyepiece under a dissecting microscope. The mean apical leakage of six groups was as follows: I = 0.45 mm, II = 0.277 mm, III = 0.343 mm, IV = 0.263 mm, V = 0.336 mm, and VI = 2.310 mm. Results showed no significant difference in the apical seal produced by gutta-percha and the tested root canal sealers.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Hydroxide↗