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ABSTRACT: The text mining and full-text searching function were used to analyze the digitalized daily reports of the Practical Training in Pharmacology submitted to the Web system in Fukuyama University. Collocations connected to the word "inspection" were searched in the daily reports using the text mining and the full-text search functions of the system. Many collocation groups connected to "inspection" were found in the daily reports and the greatest number of collocations was associated with "preparation of drugs". Practical training in narcotic dispensing has two different aspects: inspection and experience training. The number of people who reported a relation between "inspection" and "narcotics dispensing" in the daily reports was very similar to those who reported a connection between "experience" and "narcotics". Practical training to handle narcotic dispensing is the most fundamental training that the pharmacist must undertake. The progression of team-based medical care has caused medical personnel to recognize the practical training in pharmacology, and the number of the people who reported a relation between "inspection" and "team medical care" in 2011 increased in comparison to 2010. Moreover, the progression of cooperation among hospitals, pharmacies, and local blanches of the Japan Pharmaceutical Association is beneficial to practical training. Practical training in pharmacology is based on the core curriculum, but undergoes periodic modification due to societal circumstances. Therefore, these results suggest that the construction of Web system for submitting daily reports is useful for analyzing the daily reports.
YAKUGAKU ZASSHI 03/2013; · 0.37 Impact Factor
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ABSTRACT: Dipeptidyl peptidase III (DPP III), the zinc peptidase, has a unique helix portion in the metal-binding motif (HELLGH). The enzyme activity of the cupric derivative of rat DPP III (Cu(II)-rat DPP III) for Lys-Ala-β-NA is about 30% of that of the wild-type enzyme. On the other hand, the enzyme activity of Cu(II)-rat del-DPP III, in which Leu453 is deleted from the metal-binding motif, possesses only 1-2% of the enzyme activity of rat del-DPP III. The EPR spectra of Cu(II)-rat DPP III in the presence of various concentrations of the substrate, Lys-Ala-β-NA, changed dramatically, showing formation of the enzyme-metal-substrate complex. The EPR spectra of Cu(II)-rat del-DPP III did not change in the presence of excess Lys-Ala-β-NA. The deletion of Leu453 from the HELLGH motif of rat DPP III leads to a complete loss of flexibility in the ligand geometry around the cupric ions. Under the formation of the enzyme-metal-substrate complex, Glu451 of Cu(II)-rat DPP III is sufficiently able to approach the water molecule via a very different orientation from that of the resting state; however, Glu451 of Cu(II)-rat del-DPP III is not able to access the water molecule.
Archives of Biochemistry and Biophysics 06/2012; 525(1):71-81. · 2.93 Impact Factor
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ABSTRACT: Almost all naturally occurring metalloproteases are monozinc enzymes. The zinc in any number of zinc metalloproteases has been substituted by some other divalent cation. Almost all Co(II)- or Mn(II)-substituted enzymes maintain the catalytic activity of their zinc counterparts. However, in the case of Cu(II) substitution of zinc proteases, a great number of enzymes are not active, for example, thermolysin, carboxypeptidase A, endopeptidase from Lactococcus lactis, or aminopeptidase B, while some do have catalytic activity, for example, astacin (37%) and DPP III (100%). Based on structural studies of various metal-substituted enzymes, for example, thermolysin, astacin, aminopeptidase B, dipeptidyl peptidase (DPP) III, and del-DPP III, the metal coordination geometries of both active and inactive Cu(II)-substituted enzymes are shown to be the same as those of the wild-type Zn(II) enzymes. Therefore, the enzyme activity of a copper-ion-substituted zinc metalloprotease may depend on the flexibility of catalytic domain.
Journal of amino acids. 01/2011; 2011:574816.
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ABSTRACT: Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, "HELLGH," as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K(m) values or in the substrate specificity. However, the k(cat) values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k(cat) values of the mutants E507A and E512A were 2.38 and 3.88 s⁻¹ toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 x 10³ to 2 x 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.
Biochimica et Biophysica Acta 10/2010; 1804(10):2063-9. · 4.66 Impact Factor
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ABSTRACT: Timolol, a beta-blocker, has been shown to be an effective ocular hypotensive agent when used alone or with carbonic anhydrase inhibitor on ocular hypertensive or open angle glaucoma patients. The effect of timolol hemihydrate on the CO(2) hydration activities of human carbonic anhydrase (HCA) I and II and their reaction mechanisms were investigated. Timolol activates the enzyme activities of HCA I and HCA II. In HCA I and II, the enzyme kinetic results clearly showed that timolol increases the value of V(max) but does not influence the value of K(m). The enzyme kinetic method showed that timolol noncompetitively activates HCA I and II activities through the formation of a ternary complex consisting of the enzyme, the substrate, and timolol. These results indicate that timolol binds apart from the narrow cavity of the active site. AutoDocking results showed that timolol binds at the entrance of the active site cavity in a region where the proton shuttle residue, His 64, of HCA I or II, is placed. The enzyme kinetic and AutoDocking results showed that timolol might weakly bind near the proton shuttle residue, His 64, to accelerate the proton transfer rate from His 64 to the buffer components. It is known that efficient activators of carbonic anhydrase possess a bulky aromatic/heterocyclic moiety and a primary/secondary amino group in their molecular structure. Timolol has a heterocyclic moiety and a secondary amino group, which are typical structures in efficient activators of carbonic anhydrase.
Biological & Pharmaceutical Bulletin 02/2010; 33(2):301-6. · 1.66 Impact Factor
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ABSTRACT: Latanoprost, a prostaglandin F2 alpha analogue, has been shown to be an effective ocular hypotensive agent when used alone on ocular hypertensive or open angle glaucoma patients. Carbonic anhydrase (CA) inhibitors are also used to reduce ocular hypertension by decreasing aqueous humor secretion, and are given in combination with prostaglandin F2 alpha analogue. It has been shown that prostaglandin F2 alpha, Minprostin F2 alpha, has been shown to increase the carbonic anhydrase (CA) activity and blood pressure. However, the effects of latanoprost on CA have not been clarified. Therefore, we studied the effects of latanoprost free acid on human carbonic anhydrase (HCA) I and II using the stopped flow method. Latanoprost free acid inhibited the hydration activity of HCA I or II by a noncompetitive mechanism. The inhibition constants (Ki) of latanoprost free acid for HCA I and II were 0.22 and 2.3 mM, respectively. Therefore, latanoprost free acid is a weak inhibitor of HCA I or II. AutoDock simulation of the latanoprost free acid-HCA I or II complex showed that the carboxylic moiety of latanoprost free acid, which is located at the end of the molecule, binds to the zinc ion of the active site by stretching of the chain of latanoprost free acid through the narrow and deep active site cavity of HCA I or II. In the active site cavity of HCA I or II, one side is hydrophilic and the other is hydrophobic. AutoDock simulation results clearly showed that latanoprost free acids lie down on the hydrophobic sides of the active site cavities in HCA I and II. The noncompetitive inhibition mechanism and the binding mode of latanoprost free acid indicate that the behavior of latanoprost free acid is very similar to that of simple anions.
Biological & Pharmaceutical Bulletin 06/2008; 31(5):796-801. · 1.66 Impact Factor
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ABSTRACT: Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.
Biochemistry 10/2006; 45(38):11425-31. · 3.42 Impact Factor
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ABSTRACT: The NM-gamma CNDO/S program previously developed by our group was modified by the introduction of a new one-center electron repulsion integral gamma(AA)(new) approximation, namely, the gamma(AA)(new)-CNDO/S method. The value of this gamma(AA)(new) was evaluated according to the product values of the coefficient C with the gamma(AA) value proposed in our previous paper. This method using a new gamma(AA) was also found to improve the two-center electron repulsion integral gamma(AB) value with respect to the chemical softness proposed by Nishimoto and co-workers, together with the difference between HOMO and LUMO orbital energies. The results calculated by the present improved gamma(AA)(new)-CNDO/S method demonstrated that not only the calculated absorption maxima wavelengths and ionization potentials, but also the order and the assignment of orbitals coincided very well with those based on the results of experiments investigating a variety of polyenes, cyanynes, and polycyclic aromatic hydrocarbons.
CHEMICAL & PHARMACEUTICAL BULLETIN 06/2006; 54(5):646-52. · 1.59 Impact Factor
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ABSTRACT: The specific optical rotations of (R)-oxopropaline D calculated by two ab initio MO methods were +52+/-31 degrees and +61+/-29 degrees, respectively, and (+)-oxopropaline D (3) was presumed to have an R-configuration. On the basis of this theoretical result, the reaction of 1-litio-beta-carboline with (R)-glyceraldehyde acetonide followed by oxidation with MnO(2) gave (R)-oxopropaline D acetonide (4a), which was consistent with the previously synthesized (+)-oxopropaline D acetonide (4) in all respects. From the results of theoretical calculations and the experimental synthesis, we determined that natural (+)-oxopropaline D (3) has an R-configuration.
CHEMICAL & PHARMACEUTICAL BULLETIN 02/2003; 51(1):20-3. · 1.59 Impact Factor
