[A case of giant vaginal stone around a foreign body associated with vesicovaginal fistula].
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Adult↗
Biomedical subjects
S Kawauchi
Publications and source records attributed to S Kawauchi.
Targeting of chrolamphenicol acetyltransferase to human immunodeficiency virus particles via Vpr and Vpx.
Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N-terminus of HIV-1 Vpr, HIV-2 Vpr, or HIV-2 Vpx was incorporated into mature virions in a type-selective manner. By using chimeric proteins between HIV-1 Vpr and HIV-2 Vpx, we found that the N-terminal side of these proteins was mainly important for type-selective virion incorporation. The C-terminal arginine-rich region of HIV-1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV-2 Vpr and HIV-2 Vpx had no such activity. This region of HIV-1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.
Amino Acid Sequence↗
Bindability and digestibility of high-pressure-treated starch with glucoamylases from Rhizopus sp.
The effect of hydrostatic pressure on the bindability and digestibility of raw corn starch with two types of glucoamylase [EC 3.2.1.3] from Rhizopus sp., Gluc1 and Gluc2, was investigated in the range of 100 to 600 MPa. Pressurization of raw starch at 100 to 300 MPa for 1 h had no detectable effect, whereas pressurization at higher pressures than 400 MPa markedly enhanced the bindability and digestibility with Gluc1 and Gluc2, especially with Gluc2, which scarcely binds to raw starch and has much lower activity than Gluc1 towards raw starch. The binding constants Ks of Gluc1 and Gluc2 for the 400-MPa-pressurized starch reached 16 and 1.6 x 10(5) M-1, respectively, as compared with the Ks of 2.1 and 0.082 x 10(5) M-1 for raw starch. The 500- to 600-MPa-pressurized starch was digested by Gluc1 and Gluc2 at about 4.2 and 80 times higher rates, respectively, than raw starch. Thus, the high-pressure-treated starch was hardly distinguishable from high temperature (75 to 100 degrees C)-treated starch at least with respect to behavior with the enzymes, although some difference was observed between these starches by scanning electron microscopy and differential scanning calorimetry.
Calorimetry, Differential Scanning↗
Effects of caffeine and related methylxanthines on fetal mouse palates cultured in vitro.
The maxillary regions of day-12.5 ICR mouse fetuses were cultured in a chemically-defined serumless medium and the effects of methylxanthine derivatives on cultured palates were studied. Explanted palates were treated for 72 hr in vitro with 0.5-2 mM caffeine (CA), 1-2 mM theophylline (TP), or 1-2 mM theobromine (TB). Although the three compounds tested did not prevent the contact of opposing palatal shelves, palatal fusion was inhibited by CA and TP at a concentration of 2 mM, and the inhibitory effect of CA was more evident than that of TP. On the other hand, TB did not exert an inhibitory effect on palatogenesis at 2 mM. Since the in vitro toxicity of the methylxanthine compounds appeared to correlate well with their in vivo teratogenic potential, the organ culture method of fetal rodent palates should be a useful tool for screening teratogenic agents, especially those causing cleft palate.
Animals↗
Protein S Tokushima: an abnormal protein S found in a Japanese family with thrombosis.
An abnormal protein S (PS) was found in a Japanese family with a high incidence of thrombosis. The proband is a woman who was born in Tokushima Prefecture. She had superior sagittal sinus thrombosis, thrombophlebitis of the left leg, and thrombosis of the placenta. She had a normal plasma level of free PS antigen but decreased PS activity. Her mother and aunt also had thrombophlebitis of the leg, and together with four other family members also showed a normal level but decreased activity of PS. This suggests that hereditary dysfunction of PS is inherited in this family as an autosomal dominant trait. The proband's PS appears to have a slightly higher molecular weight than normal PS both in the intact and modified form, suggesting that it has a molecular defect on the carboxyl-terminal side of the thrombin-sensitive site. This abnormal PS with apparently unique characteristics was named PS Tokushima.
Adult↗
Kinetics of the hydrolysis of mixed micelles of dipalmitoyllecithin with triton X-100 catalyzed by a phospholipase A2 from the venom of Agkistrodon halys blomhoffii.
The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by the intact and the N-terminal alpha-NH2-modified phospholipases A2 (PLA2s) of Agkistrodon halys blomhoffii, was studied at 25 degrees C and ionic strength 0.1 in the presence of saturating amounts of Ca2+. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC6PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al. (1986) J. Biochem. 100, 1655-1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA2 [Dijkstra, B.W. et. al. (1981) J. Mol. Biol. 147, 97-123] and on a PLA2 of Crotalus atrox venom [Brunie, S. et al. (1985) J. Biol. Chem. 260, 9742-9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates.
1,2-Dipalmitoylphosphatidylcholine↗
Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis.
Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Role of Ca2+ in the substrate binding and catalytic functions of snake venom phospholipases A2.
Phospholipases A2 are classified into two groups, I and II, according to differences in the polypeptide-chain length and the intramolecular-disulfide bondings. The effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed and micellar phosphatidylcholines, catalyzed by a cobra (Naja naja atra) enzyme (Group I) and by mamushi (Agkistrodon halys blomhoffii) and habu (Trimeresurus flavoviridis) enzymes (Group II), were studied by the pH-statassay method at 25 degrees C, pH 8.0-8.2, and ionic strength 0.1-0.2. The results were compared with those reported for the other Group I and II enzymes. The Ca2+ binding was clearly shown to be essential for the catalysis of all the phospholipases A2. However, the substrate binding to Group I enzymes was found to be independent of the Ca2+ binding. On the other hand, the substrate binding to Group II enzymes was facilitated more than 10 times by the binding of Ca2+ to the enzymes. This was interpreted in terms of conformation changes of the peptide loop of residues 26 to 44 accompanying the Ca2+ binding. The latter result, but not the former, seems compatible with the hypothesis for interpreting the catalytic mechanism of phospholipases A2 that an intermediate complex should be stabilized by the coordination of the bound Ca2+ ion with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. According to the similarity in the primary and tertiary structures of the active sites of both types of enzymes [Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634], it is supposed that similar intermediate complexes may occur even for Group I enzymes, at least in the transition state of the productive complexes.
Animals↗
Pleomorphic adenoma with translocation (3;8)(p24;q13) in a child.
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Adenoma, Pleomorphic↗
Prothrombin Tokushima, a replacement of arginine-418 by tryptophan that impairs the fibrinogen clotting activity of derived thrombin Tokushima.
Structural studies on a hereditarily abnormal prothrombin, prothrombin Tokushima, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity upon conversion to thrombin. The prothrombin sample used was from a heterozygote but contained exclusively a defective prothrombin molecule, since the patient was heterozygous for both dysprothrombinemia and hypoprothrombinemia. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of the abnormal thrombin indicated that Arg-418 (equivalent to Asn-101 in the chymotrypsin numbering system) had been replaced by Trp. This amino acid substitution can result from a single nucleotide change in the codon for Arg-418 (CGG----TGG). The Arg----Trp replacement found in the thrombin portion of prothrombin Tokushima appears to reduce its interaction with various substrates including fibrinogen and platelet receptors and accounts for the recurrent bleeding episode observed in the propositus.
Amino Acid Sequence↗
Determination of binding constants for N-acetyl-D-glucosamine oligomers with lysozyme.
A method to determine intrinsic binding constants of lysozyme with substrate analogues such as N-acetyl-D-glucosamine dimer and trimer is proposed. The method is based on the competitive interaction of an anionic azo dye with substrate analogues for lysozyme. There are two binding sites for substrate analogues and dyes, respectively, on lysozyme. One binding mode of the substrate analogues to subsites D-F on lysozyme was non-competitive, and another binding mode to subsites A-C was competitive with the dye. From the binding constants obtained it is suggested that the binding of the substrate analogues to subsite D on lysozyme is weaker than the binding to the other subsites.
Acetylglucosamine↗
[Dysprothrombinemia--with special reference to prothrombin Tokushima].
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Adolescent↗
Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin.
A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of "prothrombin" by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of "prothrombin." Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of "thrombin." Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of "thrombin" when Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p'-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as "thrombin" in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than "thrombin" when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as "thrombin" in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.
Blood Coagulation Disorders↗
Plasma fibronectin deficiency in eight members of one family.
The proband, a 31-year-old female, had keloids at the sites of surgery and burns. Her plasma fibronectin level measured by Laurell's method was only 10 mg/dl (mean +/- SD in 101 healthy controls 32.2 +/- 5.6 mg/dl) but the electrophoretic mobility was normal. Low levels of plasma fibronectin (range 11-17 mg/dl) were found in the proband's mother, two uncles, two brothers, one daughter, and one nephew. Levels in her father and other family members examined were normal (range 32-51 mg/dl). The proband has been quite healthy, an haematological and haemostatic tests showed no abnormality except reduced phagocytosis by neutrophils. Other family members with low plasma fibronectin levels had no abnormalities on examination and no keloids.
Adolescent↗
Changes in plasma fibronectin levels in thyroid diseases.
The plasma levels of fibronectin (Fn) have been measured in normal subjects and in patients with thyroid diseases. The mean plasma Fn levels in 62 normal adults was 32.0 +/- 6.0 mg/dl, whereas it was elevated to 62.6 +/- 16.1 mg/dl (mean +/- SD) in 25 patients with hyperthyroidism and decreased to 19.2 +/- 8.0 mg/dl in 9 patients with hypothyroidism. The 9 patients with simple goiter have normal values of 29.1 +/- 8.0 mg/dl. With the administration of anti-thyroid drugs, plasma Fn levels normalized, with a time lag, in parallel with normalization of the thyroid function. Positive correlation was obtained between Fn levels and serum levels of triiodothyronine (T3) and thyroxine (T4). The present findings indicate that measurement of plasma Fn both in the basal state and during treatment provides evidence of altered Fn metabolism in thyroid diseases and serves to follow up the effect of treatment.
Adolescent↗
Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by the phospholipase A2 from Agkistrodon halys blomhoffii venom.
The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the phospholipase A2 from the venom of Agkistrodon halys blomhoffii, was studied at 25 degrees C and the ionic strength of 0.1 in the presence of 3-33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the alpha-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799-804, and Haruki et al. (1986) J. Biochem. 99, 99-109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125-1130).(ABSTRACT TRUNCATED AT 250 WORDS)
Catalysis↗
Interaction of monodispersed and micellar phospholipids with an Agkistrodon halys blomhoffii phospholipase A2, in which the alpha-amino group had been modified to an alpha-keto group.
The pH dependence of the binding constant of Ca2+ to a phospholipase A2 of Agkistrodon halys blomhoffii, in which the alpha-amino group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 by the tryptophyl fluorescence method. The dependence was compared with the results for the intact enzyme (Ikeda et al. (1981) J. Biochem. 90, 1125-1130). The pH-dependence curve could be well interpreted in terms of the participation of the two ionizable groups Asp 49 and His 48, with pK values of 4.70 and 6.69, respectively. These values were slightly different from the respective pK values for the intact enzyme, 5.15 and 6.45. Ca2+ binding to the intact enzyme involves the participation of an additional ionizable group with a pK value of 7.30, which was thus assigned as alpha-amino group. The pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) method. The pH-dependence curve for the modified apoenzyme was interpreted as reflecting the participation of a single ionizable group with a pK value of 4.7, which was assigned to Asp 49 (to which a Ca2+ ion can coordinate) since the curve for the Ca2+ complex lacked this transition: the binding constant was independent of pH. The pH-dependence curves for the intact apoenzyme and its Ca2+ complex involve the participation of an additional ionizable group with pK values of 7.30 and 6.30, respectively (Ikeda & Samejima (1981) J. Biochem. 90, 799-804), which was assigned as the alpha-amino group. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the intact and the alpha-NH2-modified enzymes was studied by the pH stat method at 25 degrees C, pH 8.2, and ionic strength 0.1 in the presence of 3 mM Ca2+. The Km value for the modified enzyme was found to be very similar to that for the intact enzyme: this was compatible with the results of the direct binding study on the monodispersed n-C12PC under the same conditions. However, the kcat value was about 43% of the value for the intact enzyme, suggesting that the alpha-keto group introduced by the chemical modification perturbed the network of hydrogen bonds in the active site.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acids↗
Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2.
The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A2, was studied at 25 degrees C ionic strength 0.1 in the presence of 3-10 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C12PC). These results indicated that the substrate and n-C12PC compete for the same site on the enzyme molecule. The pH dependence curve of the kinetic parameter, kcat/Km, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20).(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Sites↗