Ito K

References (45)

Title : Network-based meta-analysis and the candidate gene association studies reveal novel ethnicity-specific variants in MFSD3 and MRPL43 associated with dementia with Lewy bodies - Shigemizu_2022_Am.J.Med.Genet.B.Neuropsychiatr.Genet__
Author(s) : Shigemizu D , Asanomi Y , Akiyama S , Higaki S , Sakurai T , Ito K , Niida S , Ozaki K
Ref : American Journal of Medicine Genet B Neuropsychiatr Genet , : , 2022
Abstract : Dementia with Lewy bodies (DLB) is the second most common form of neurodegenerative dementia in elderly people, following Alzheimer's disease. Only three genes, SNCA (alpha-synuclein), APOE (apolipoprotein E), and GBA (glucosylceramidase), have been convincingly demonstrated to be associated with DLB. Here, we applied whole-genome sequencing to blood samples from 61 DLB patients and 45 cognitively normal controls. We used accumulation of candidate mutations to detect novel DLB-associated genes. Subsequent single nucleotide polymorphism (SNP) genotyping and association studies in a large number of samples from Japanese individuals revealed novel heterozygous variants in MFSD3 (rs143475431, c.888T>A:p.C296*; n = 5,421, p = 0.00063) and MRPL43 (chr10:102746730, c.241A>C:p.N81H; n = 4,782, p = 0.0029). We further found that the MFSD3 variant increased plasma levels of butyrylcholinesterase (n = 1,206, p = 0.029). We believe that our findings will contribute to the understanding of DLB and provide insight into its pathogenic mechanism for future studies.
ESTHER : Shigemizu_2022_Am.J.Med.Genet.B.Neuropsychiatr.Genet__
PubMedSearch : Shigemizu_2022_Am.J.Med.Genet.B.Neuropsychiatr.Genet__
PubMedID: 35765761

Title : Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research - Yamashita_2021_Mol.Ther.Methods.Clin.Dev_22_263
Author(s) : Yamashita T , Inui T , Yokota J , Kawakami K , Morinaga G , Takatani M , Hirayama D , Nomoto R , Ito K , Cui Y , Ruez S , Harada K , Kishimoto W , Nakase H , Mizuguchi H
Ref : Mol Ther Methods Clin Dev , 22 :263 , 2021
Abstract : The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.
ESTHER : Yamashita_2021_Mol.Ther.Methods.Clin.Dev_22_263
PubMedSearch : Yamashita_2021_Mol.Ther.Methods.Clin.Dev_22_263
PubMedID: 34485610

Title : Hydrophobic interactions at subsite S1' of human dipeptidyl peptidase IV contribute significantly to the inhibitory effect of tripeptides - Araki_2020_Heliyon_6_e04227
Author(s) : Araki M , Kanegawa N , Iwata H , Sagae Y , Ito K , Masuda K , Okuno Y
Ref : Heliyon , 6 :e04227 , 2020
Abstract : Functional inhibitory peptides of human dipeptidyl peptidase 4 (hDPP4) have been highly anticipated as the active ingredient of functional food for type II diabetes; however, the molecular mechanism of hDPP4 inhibition remains unclear. In this study, we focused on dipeptides and tripeptides, which display structure-function correlations that are relatively easy to analyze, and examined their interactions with hDPP4 on an atomic level using a combination of docking studies and an hDPP4 inhibition assay. First, we performed comprehensive binding mode analysis of the dipeptide library and demonstrated that the formation of a tight interaction with the S1 subsite composing part of the substrate pocket is essential for dipeptides to compete with the substrate and strongly inhibit hDPP4. Next, we synthesized tripeptides by adding various amino acids to the C-terminus of Ile-Pro and Val-Pro, which have especially high inhibitory activity among compounds in the dipeptide library, and measured the hDPP4 inhibitory activity of the tripeptides. When hydrophobic amino acids (Ile, Met, Val, Trp) were added, the inhibitory activity increased several-fold. This phenomenon could be explained as follows: the C-terminal amino acid of the tripeptide formed hydrophobic interactions with Tyr547 and Trp629, which compose the S1' subsite located relatively outside the substrate pocket, thereby stabilizing the hDPP4-peptide binding. The structural information on the interaction between hDPP4 and peptide inhibitors attained in this study is anticipated to be useful in the development of a more potent hDPP4 competitive inhibitor.
ESTHER : Araki_2020_Heliyon_6_e04227
PubMedSearch : Araki_2020_Heliyon_6_e04227
PubMedID: 32613113

Title : Differentiation Between Dementia With Lewy Bodies And Alzheimer's Disease Using Voxel-Based Morphometry Of Structural MRI: A Multicenter Study - Matsuda_2019_Neuropsychiatr.Dis.Treat_15_2715
Author(s) : Matsuda H , Yokoyama K , Sato N , Ito K , Nemoto K , Oba H , Hanyu H , Kanetaka H , Mizumura S , Kitamura S , Shinotoh H , Shimada H , Suhara T , Terada H , Nakatsuka T , Kawakatsu S , Hayashi H , Asada T , Ono T , Goto T , Shigemori K
Ref : Neuropsychiatr Dis Treat , 15 :2715 , 2019
Abstract : Background: The differential diagnosis of dementia with Lewy bodies (DLB) and Alzheimer's disease (AD) is particularly important because DLB patients respond better to cholinesterase inhibitors but sometimes exhibit sensitivity to neuroleptics, which may cause worsening of clinical status. Antemortem voxel-based morphometry (VBM) using structural MRI has previously revealed that patients with DLB have normal hippocampal volume, but atrophy in the dorsal mesopontine area. Objectives: The aim of this multicenter study was to determine whether VBM of the brain stem in addition to that of medial temporal lobe structures improves the differential diagnosis of AD and DLB. Methods: We retrospectively chose 624 patients who were clinically diagnosed with either DLB (239 patients) or AD (385 patients) from 10 institutes using different MR scanners with different magnetic field strengths. In all cases, VBM was performed on 3D T1-weighted images. The degree of local atrophy was calculated using Z-score by comparison with a database of normal volumes of interest (VOIs) in medial temporal lobe (MTL) and the dorsal brain stem (DBS). The discrimination of DLB and AD was evaluated using Z-score values in these two VOIs. MRI data from 414 patients were used as the training data set to determine the classification criteria, with the MRI data from the remaining 210 patients used as the test data set. Results: The DLB and AD patients did not differ with respect to mean age or Mini-Mental State Examination scores. Z-index scores showed that there was significantly more atrophy in MTL of AD patients, compared to DLB patients and in DBS of DLB patients, compared to AD patients. The discrimination accuracies of VBM were 63.3% in the test data set and 73.4% in the training data set. Conclusion: VBM of DBS in addition to that of MTL improves the differentiation of DLB and AD.
ESTHER : Matsuda_2019_Neuropsychiatr.Dis.Treat_15_2715
PubMedSearch : Matsuda_2019_Neuropsychiatr.Dis.Treat_15_2715
PubMedID: 31571887

Title : Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum - Araki_2019_Proc.Natl.Acad.Sci.U.S.A_116_8269
Author(s) : Araki Y , Awakawa T , Matsuzaki M , Cho R , Matsuda Y , Hoshino S , Shinohara Y , Yamamoto M , Kido Y , Inaoka DK , Nagamune K , Ito K , Abe I , Kita K
Ref : Proc Natl Acad Sci U S A , 116 :8269 , 2019
Abstract : Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.
ESTHER : Araki_2019_Proc.Natl.Acad.Sci.U.S.A_116_8269
PubMedSearch : Araki_2019_Proc.Natl.Acad.Sci.U.S.A_116_8269
PubMedID: 30952781
Gene_locus related to this paper: acreg-ascc

Title : Predictors of Postoperative Non-Chylous Massive Discharge after Pancreaticoduodenectomy for Pancreatic Ductal Adenocarcinoma - Ito_2018_Dig.Surg_35_252
Author(s) : Ito K , Kawaguchi Y , Sakamoto Y , Arita J , Hasegawa K , Kokudo N
Ref : Dig Surg , 35 :252 , 2018
Abstract : BACKGROUND: Pancreaticoduodenectomy (PD) is performed for pancreatic ductal adenocarcinoma (PDA) located at the pancreas head/body. Non-chylous massive discharge after PD is frequently encountered and can cause a vicious cycle of complications associated with severe dehydration and protein loss. METHODS: From August 2008 to June 2015, 102 patients who underwent PD for PDA were retrospectively reviewed. High non-chylous discharge was defined as postoperative daily serous discharge exceeding 10 mL/kg. Predictive factors for high non-chylous discharge were assessed using multivariable analysis. RESULTS: Fifty-one patients (50%) developed high non-chylous discharge. Body mass index (BMI) and hemoglobin, total protein, and cholinesterase levels were significantly lower in the high-discharge group compared to the corresponding levels in the low-discharge group. The incidence of postoperative pancreatic fistula and delayed gastric emptying was significantly lower and higher in the high-discharge group than in the low-discharge group, respectively. Multivariable analysis revealed that BMI <22.0 kg/m2, hemoglobin <12.0 g/dL and intraoperative blood loss >/=800 mL were independent predictive factors for high non-chylous discharge. CONCLUSIONS: Preoperative low levels of BMI and hemoglobin and intraoperative high blood loss were independent predictive factors for high non-chylous discharge. Improvement of preoperative general and nutritional condition may reduce the incidence of high non-chylous discharge.
ESTHER : Ito_2018_Dig.Surg_35_252
PubMedSearch : Ito_2018_Dig.Surg_35_252
PubMedID: 28817822

Title : Acotiamide Hydrochloride, a Therapeutic Agent for Functional Dyspepsia, Enhances Acetylcholine-induced Contraction via Inhibition of Acetylcholinesterase Activity in Circular Muscle Strips of Guinea Pig Stomach - Ito_2016_Drug.Res.(Stuttg)_66_196
Author(s) : Ito K , Kawachi M , Matsunaga Y , Hori Y , Ozaki T , Nagahama K , Hirayama M , Kawabata Y , Shiraishi Y , Takei M , Tanaka T
Ref : Drug Res (Stuttg) , 66 :196 , 2016
Abstract : Acotiamide is a first-in-class prokinetic drug approved in Japan for the treatment of functional dyspepsia. Given that acotiamide enhances gastric motility in conscious dogs and rats, we assessed the in vitro effects of this drug on the contraction of guinea pig stomach strips and on acetylcholinesterase (AChE) activity in stomach homogenate following fundus removal. We also investigated the serotonin 5-HT4 receptor agonist mosapride, dopamine D2 receptor and AChE inhibitor itopride, and representative AChE inhibitor neostigmine. Acotiamide (0.3 and 1 muM) and itopride (1 and 3 muM) significantly enhanced the contraction of gastric body strips induced by electrical field stimulation (EFS), but mosapride (1 and 10 muM) did not. Acotiamide and itopride significantly enhanced the contraction of gastric body and antrum strips induced by acetylcholine (ACh), but not that induced by carbachol (CCh). Neostigmine also significantly enhanced the contraction of gastric body strips induced by ACh, but not that by CCh. In contrast, mosapride failed to enhance contractions induced by either ACh or CCh in gastric antrum strips. Acotiamide exerted mixed inhibition of AChE, and the percentage inhibition of acotiamide (100 muM) against AChE activity was markedly reduced after the reaction mixture was dialyzed. In contrast, itopride exerted noncompetitive inhibition on AChE activity. These results indicate that acotiamide enhances ACh-dependent contraction in gastric strips of guinea pigs via the inhibition of AChE activity, and that it exerts mixed and reversible inhibition of AChE derived from guinea pig stomach.
ESTHER : Ito_2016_Drug.Res.(Stuttg)_66_196
PubMedSearch : Ito_2016_Drug.Res.(Stuttg)_66_196
PubMedID: 26418413

Title : Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor - Lan_2015_Food.Chem_175_66
Author(s) : Lan VT , Ito K , Ohno M , Motoyama T , Ito S , Kawarasaki Y
Ref : Food Chem , 175 :66 , 2015
Abstract : Human dipeptidyl peptidase IV (hDPPIV) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Because certain peptides are known to act as hDPPIV inhibitors, a dataset of possible peptides with their inhibition intensities will facilitate the development of functional food for type 2 diabetes. In this study, we examined a total of 337 dipeptides with respect to their hDPPIV inhibitory effects. Amino acid residues at N-termini dominated their inhibition intensities. Particularly highly inhibitory dipeptides discovered included the following novel dipeptides: Thr-His, Asn-His, Val-Leu, Met-Leu, and Met-Met. Using our dataset, prime candidates contributing to the hDPPIV inhibitory effect of soy protein hydrolyzates were successfully identified. Possible dietary proteins potentially able to produce particularly highly hDPPIV inhibitory peptides are also discussed on the basis of the dataset.
ESTHER : Lan_2015_Food.Chem_175_66
PubMedSearch : Lan_2015_Food.Chem_175_66
PubMedID: 25577052

Title : Trp-Arg-Xaa tripeptides act as uncompetitive-type inhibitors of human dipeptidyl peptidase IV - Lan_2014_Peptides_54C_166
Author(s) : Lan VT , Ito K , Ito S , Kawarasaki Y
Ref : Peptides , 54C :166 , 2014
Abstract : Human dipeptidyl peptidase IV (hDPPIV, alternative name: CD26) inhibitors provide an effective strategy for the treatment of type 2 diabetes. Recently, our research group discovered a non substrate-mimic inhibitory dipeptide, Trp-Arg, by the systematic analysis of a dipeptide library. In the present study, a tripeptide library Trp-Arg-Xaa (where Xaa represents any amino acid) was analyzed to investigate the interactions of peptidergic inhibitors with hDPPIV. Trp-Arg-Glu showed the highest inhibitory effect toward hDPPIV (Ki=130muM). All of the tested 19 Trp-Arg-Xaa tripeptides showed unique uncompetitive-type inhibition. The inhibition mechanism of Trp-Arg-Xaa is discussed based on the crystal structure of hDPPIV. The information obtained by this study suggests a novel concept for developing hDPPIV inhibitory peptides and drugs.
ESTHER : Lan_2014_Peptides_54C_166
PubMedSearch : Lan_2014_Peptides_54C_166
PubMedID: 24512990

Title : Systematic analysis of a dipeptide library for inhibitor development using human dipeptidyl peptidase IV produced by a Saccharomyces cerevisiae expression system - Hikida_2013_Biochem.Biophys.Res.Commun_430_1217
Author(s) : Hikida A , Ito K , Motoyama T , Kato R , Kawarasaki Y
Ref : Biochemical & Biophysical Research Communications , 430 :1217 , 2013
Abstract : The inhibition of human dipeptidyl peptidase IV/CD26 (hDPPIV) is an accepted treatment for type 2 diabetes. In this study, an extracellular production system of hDPPIV using Saccharomyces cerevisiae was established to facilitate the screening of hDPPIV inhibitors. As dipeptides that mimic the hDPPIV substrate are candidate inhibitors of this protein, X-Ala or X-Pro dipeptides (in which X represents any amino acid) were tested systematically. Based on the results obtained in the first screening, a second screening was performed for Trp-X dipeptides. To elucidate the manner via which the physicochemical features at the P(1) and P(2) positions contributed to the hDPPIV inhibitory effect, correlations between the inhibitory activity of dipeptides and 13 amino acid indices were analyzed. The most effective inhibitory dipeptide was Trp-Pro (K(i)=0.04 mM). The mode of inhibition of hDPPIV by dipeptides was explained well by some amino acid indices and by the structure of the substrate-binding site of hDPPIV. The information obtained from the systematic analysis of a dipeptide library provides important clues for the development of hDPPIV targeting drugs and functional foods for type 2 diabetes.
ESTHER : Hikida_2013_Biochem.Biophys.Res.Commun_430_1217
PubMedSearch : Hikida_2013_Biochem.Biophys.Res.Commun_430_1217
PubMedID: 23268343

Title : Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) - Ming_2013_Genome.Biol_14_R41
Author(s) : Ming R , VanBuren R , Liu Y , Yang M , Han Y , Li LT , Zhang Q , Kim MJ , Schatz MC , Campbell M , Li J , Bowers JE , Tang H , Lyons E , Ferguson AA , Narzisi G , Nelson DR , Blaby-Haas CE , Gschwend AR , Jiao Y , Der JP , Zeng F , Han J , Min XJ , Hudson KA , Singh R , Grennan AK , Karpowicz SJ , Watling JR , Ito K , Robinson SA , Hudson ME , Yu Q , Mockler TC , Carroll A , Zheng Y , Sunkar R , Jia R , Chen N , Arro J , Wai CM , Wafula E , Spence A , Xu L , Zhang J , Peery R , Haus MJ , Xiong W , Walsh JA , Wu J , Wang ML , Zhu YJ , Paull RE , Britt AB , Du C , Downie SR , Schuler MA , Michael TP , Long SP , Ort DR , Schopf JW , Gang DR , Jiang N , Yandell M , dePamphilis CW , Merchant SS , Paterson AH , Buchanan BB , Li S , Shen-Miller J
Ref : Genome Biol , 14 :R41 , 2013
Abstract : BACKGROUND: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. RESULTS: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101x and 5.2x. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. CONCLUSIONS: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.
ESTHER : Ming_2013_Genome.Biol_14_R41
PubMedSearch : Ming_2013_Genome.Biol_14_R41
PubMedID: 23663246
Gene_locus related to this paper: nelnu-a0a1u8aj84 , nelnu-a0a1u8bpe4 , nelnu-a0a1u7z9m9 , nelnu-a0a1u7ywy5 , nelnu-a0a1u8aik2 , nelnu-a0a1u7zmb5 , nelnu-a0a1u8a7m7 , nelnu-a0a1u8b0n9 , nelnu-a0a1u8b461 , nelnu-a0a1u7zzj3 , nelnu-a0a1u8ave7 , nelnu-a0a1u7yn26

Title : Potential clinical factors affecting hepatobiliary enhancement at Gd-EOB-DTPA-enhanced MR imaging - Higaki_2012_Magn.Reson.Imaging_30_689
Author(s) : Higaki A , Tamada T , Sone T , Kanki A , Sato T , Tanimoto D , Higashi H , Ito K
Ref : Magn Reson Imaging , 30 :689 , 2012
Abstract : OBJECTIVE: The objective was to clarify the clinical factors that might affect the degree of hepatic parenchymal enhancement at gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: A total of 84 patients with (n=63) and without chronic liver disease (n=21) underwent Gd-EOB-DTPA-enhanced MR imaging. Contrast-enhanced MR images of hepatobiliary phase (HP) were obtained at 20 min after Gd-EOB-DTPA administration. The relative enhancement (RE) of liver parenchyma at 20 min HP was calculated from region of interest measurements at each patient. Then, these results were correlated with various clinical parameters using Pearson correlation coefficient or Spearman rank correlation coefficient. Furthermore, the predictor of the degree of hepatic parenchymal enhancement was determined using multiple regression analysis. RESULTS: The presence or absence of chronic liver disease (P=.002), ascites (P=.005) and splenomegaly (P=.027), and the values of prothrombin activity (P=.008), total bilirubin (T-Bil) (P=.001), albumin (P=.001), aspartate aminotransferase (AST) (P=.002) and cholinesterase (P=.007) were significantly correlated with the RE of liver parenchyma at 20 min HP. Among these parameters, increases of T-Bil (P=.011 to .028) and AST (P=.018 to .049) were predictors of decreased hepatic parenchymal enhancement. CONCLUSIONS: Hepatic parenchymal enhancement of Gd-EOB-DTPA was affected by various clinical parameters. Impaired hepatobiliary enhancement may be predicted by routine biochemical tests, such as T-Bil and AST.
ESTHER : Higaki_2012_Magn.Reson.Imaging_30_689
PubMedSearch : Higaki_2012_Magn.Reson.Imaging_30_689
PubMedID: 22459437

Title : Combining patient-level and summary-level data for Alzheimer's disease modeling and simulation: a beta regression meta-analysis - Rogers_2012_J.Pharmacokinet.Pharmacodyn_39_479
Author(s) : Rogers JA , Polhamus D , Gillespie WR , Ito K , Romero K , Qiu R , Stephenson D , Gastonguay MR , Corrigan B
Ref : J Pharmacokinet Pharmacodyn , 39 :479 , 2012
Abstract : Our objective was to develop a beta regression (BR) model to describe the longitudinal progression of the 11 item Alzheimer's disease (AD) assessment scale cognitive subscale (ADAS-cog) in AD patients in both natural history and randomized clinical trial settings, utilizing both individual patient and summary level literature data. Patient data from the coalition against major diseases database (3,223 patients), the Alzheimer's disease neruroimaging initiative study database (186 patients), and summary data from 73 literature references (representing 17,235 patients) were fit to a BR drug-disease-trial model. Treatment effects for currently available acetyl cholinesterase inhibitors, longitudinal changes in disease severity, dropout rate, placebo effect, and factors influencing these parameters were estimated in the model. Based on predictive checks and external validation, an adequate BR meta-analysis model for ADAS-cog using both summary-level and patient-level data was developed. Baseline ADAS-cog was estimated from baseline MMSE score. Disease progression was dependent on time, ApoE4 status, age, and gender. Study drop out was a function of time, baseline age, and baseline MMSE. The use of the BR constrained simulations to the 0-70 range of the ADAS-cog, even when residuals were incorporated. The model allows for simultaneous fitting of summary and patient level data, allowing for integration of all information available. A further advantage of the BR model is that it constrains values to the range of the original instrument for simulation purposes, in contrast to methodologies that provide appropriate constraints only for conditional expectations.
ESTHER : Rogers_2012_J.Pharmacokinet.Pharmacodyn_39_479
PubMedSearch : Rogers_2012_J.Pharmacokinet.Pharmacodyn_39_479
PubMedID: 22821139

Title : Acotiamide, a new orally active acetylcholinesterase inhibitor, stimulates gastrointestinal motor activity in conscious dogs - Nagahama_2012_Neurogastroenterol.Motil_24_566
Author(s) : Nagahama K , Matsunaga Y , Kawachi M , Ito K , Tanaka T , Hori Y , Oka H , Takei M
Ref : Neurogastroenterol Motil , 24 :566 , 2012
Abstract : UNLABELLED: BACKGROUND Acotiamide hydrochloride (acotiamide), a novel selective acetylcholinesterase (AChE) inhibitor, has proven significantly effective in treating functional dyspepsia (FD) in clinical trials, particularly in alleviating meal-related symptoms. In the present study, we examined the gastrointestinal prokinetic effects of acotiamide administered orally or intraduodenally in conscious dogs and investigated in vivo and ex vivo anti-AChE activity of acotiamide to clarify its mechanism of prokinetic action. METHODS: Gastrointestinal motility was measured in conscious dogs with chronically implanted force transducers. KEY RESULTS: Oral administration of acotiamide stimulated postprandial gastroduodenal and colonic motor activities. Measurement of gastrointestinal motility showed that acotiamide, like itopride and mosapride, enhanced gastric antral motility. Further, acotiamide markedly improved clonidine (an alpha(2) -adrenoceptor agonist)-induced hypomotility in a dog model of gastric motor dysfunction. The postprandial gastric antral motility enhanced by acotiamide was completely abolished on treatment with the muscarinic receptor antagonist atropine. Results of an in vivo experiment on anti-AChE activity showed clearly increased acetylcholine-induced gastric motility on intraduodenal administration of acotiamide, just as observed with the AChE inhibitor neostigmine. Further, in ex vivo experiment, intraduodenal administration of acotiamide significantly inhibited AChE activity in canine gastric antrum. CONCLUSIONS & INFERENCES: Our findings revealed that acotiamide administered through the alimentary tract exerts gastroprokinetic action via cholinergic pathways by inhibiting AChE activity. These results may also confirm the mechanism of action in clinical efficacy of acotiamide on FD.
ESTHER : Nagahama_2012_Neurogastroenterol.Motil_24_566
PubMedSearch : Nagahama_2012_Neurogastroenterol.Motil_24_566
PubMedID: 22429221

Title : Acotiamide hydrochloride (Z-338) enhances gastric motility and emptying by inhibiting acetylcholinesterase activity in rats - Kawachi_2011_Eur.J.Pharmacol_666_218
Author(s) : Kawachi M , Matsunaga Y , Tanaka T , Hori Y , Ito K , Nagahama K , Ozaki T , Inoue N , Toda R , Yoshii K , Hirayama M , Kawabata Y , Takei M
Ref : European Journal of Pharmacology , 666 :218 , 2011
Abstract : In clinical trials, acotiamide hydrochloride (acotiamide: Z-338) has been reported to be useful in the treatment of functional dyspepsia. Here, we investigated the effects of acotiamide on gastric contraction and emptying activities in rats in comparison with itopride hydrochloride (itopride) and mosapride citrate (mosapride). We also examined in vitro the compound's inhibitory effect on acetylcholinesterase (AChE) activity derived from rat stomach. In in vivo studies, acotiamide (30 and 100mg/kg s.c.) and itopride (100mg/kg s.c.) markedly enhanced normal gastric antral motility in rats. In gastric motility dysfunction models, acotiamide (100mg/kg s.c.) and itopride (100mg/kg s.c.) improved both gastric antral hypomotility and the delayed gastric emptying induced by clonidine, an alpha(2)-adrenoceptor agonist. In contrast, mosapride (10mg/kg s.c.) had no effect on these models. Like the AChE inhibitors itopride (30 mg/kg s.c.) and neostigmine (10 mug/kg s.c.), acotiamide (10mg/kg s.c.) also clearly enhanced gastric body contractions induced by electrical stimulation of the vagus, which were abolished by atropine and hexamethonium, whereas mosapride (3 and 10mg/kg s.c.) did not. In in vitro studies, acotiamide concentration-dependently inhibited rat stomach-derived AChE activity (IC(50)=2.3 mumol/l). In addition, stomach tissue concentrations of acotiamide after administration at 10mg/kg s.c. were sufficient to produce inhibition of AChE activity in rat stomach. These results suggest that acotiamide stimulates gastric motility and improves gastric motility dysfunction in rats by inhibiting AChE activity, and may suggest a role for acotiamide in improving gastric motility dysfunction in patients with functional dyspepsia.
ESTHER : Kawachi_2011_Eur.J.Pharmacol_666_218
PubMedSearch : Kawachi_2011_Eur.J.Pharmacol_666_218
PubMedID: 21651906

Title : Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis - Shinkuma_2010_Hum.Mutat_31_602
Author(s) : Shinkuma S , Akiyama M , Inoue A , Aoki J , Natsuga K , Nomura T , Arita K , Abe R , Ito K , Nakamura H , Ujiie H , Shibaki A , Suga H , Tsunemi Y , Nishie W , Shimizu H
Ref : Hum Mutat , 31 :602 , 2010
Abstract : Autosomal recessive hypotrichosis (ARH) is characterized by sparse hair on the scalp without other abnormalities. Three genes, DSG4, LIPH, and LPAR6 (P2RY5), have been reported to underlie ARH. We performed a mutation search for the three candidate genes in five independent Japanese ARH families and identified two LIPH mutations: c.736T>A (p.Cys246Ser) in all five families, and c.742C>A (p.His248Asn) in four of the five families. Out of 200 unrelated control alleles, we detected c.736T>A in three alleles and c.742C>A in one allele. Haplotype analysis revealed each of the two mutant alleles is derived from a respective founder. These results suggest the LIPH mutations are prevalent founder mutations for ARH in the Japanese population. LIPH encodes PA-PLA(1)alpha (LIPH), a membrane-associated phosphatidic acid-preferring phospholipase A(1)alpha. Two residues, altered by these mutations, are conserved among PA-PLA(1)alpha of diverse species. Cys(246) forms intramolecular disulfide bonds on the lid domain, a crucial structure for substrate recognition, and His(248) is one amino acid of the catalytic triad. Both p.Cys246Ser- and p.His248Asn-PA-PLA(1)alpha mutants showed complete abolition of hydrolytic activity and had no P2Y5 activation ability. These results suggest defective activation of P2Y5 due to reduced 2-acyl lysophosphatidic acid production by the mutant PA-PLA(1)alpha is involved in the pathogenesis of ARH.
ESTHER : Shinkuma_2010_Hum.Mutat_31_602
PubMedSearch : Shinkuma_2010_Hum.Mutat_31_602
PubMedID: 20213768
Gene_locus related to this paper: human-LIPH

Title : Disease progression meta-analysis model in Alzheimer's disease - Ito_2010_Alzheimers.Dement_6_39
Author(s) : Ito K , Ahadieh S , Corrigan B , French J , Fullerton T , Tensfeldt T
Ref : Alzheimers Dement , 6 :39 , 2010
Abstract : BACKGROUND: Various authors have evaluated disease progression in Alzheimer's disease (AD), using patient data from individual clinical studies or pooled data across various trials. We conducted a systematic review of public data sources from 1990 to 2008 for all available AChE inhibitor studies, as well as clinical studies that evaluated the rate of deterioration in AD patients. Unique to this analysis, we developed a model based on literature data to describe the longitudinal response in the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-cog) (change from baseline) in mild to moderate severity AD patients. The model was used to estimate disease progression for both placebo-treated patients and acetylcholinesterase (AChE)-inhibitor treated patients, and factors that affected disease progression. METHODS: We collected 576 mean ADAS-cog changes from baseline data points of 52 trials, representing data from approximately 19,972 patients and more than 84,000 individual observations. The model described the rate of disease progression, the evident placebo effect, and the symptomatic effect of AChE-inhibitors. Baseline ADAS-cog, Mini-Mental State Examination score, age, and year of publication were tested as covariates. RESULTS: The disease progression in mild to moderate AD patients across all available and relevant literature sources was estimated as 5.5 points per year. An Emax-type model best described the symptomatic drug effect of AChE inhibitors. The rate of disease progression (underlying disease progression) was no different between placebo and AChE-inhibitors groups. Baseline ADAS-cog is a significant covariate in disease progression. Baseline age was also tested as a covariate in the rate of disease progression, but the model was unable to describe any effects of age, likely because of the narrow distribution of mean age (literature-level analysis). There was no significant impact of publication year in the model. CONCLUSIONS: Baseline ADAS-cog is a significant covariate affecting the rate of disease progression, and it describes or at least explains the different rates of deterioration evident in early or late stages of the disease. There was no significant impact of publication year in the model, suggesting that disease progression has not slowed in more recent trials.
ESTHER : Ito_2010_Alzheimers.Dement_6_39
PubMedSearch : Ito_2010_Alzheimers.Dement_6_39
PubMedID: 19592311

Title : Characterization of inhibitory effect of carbapenem antibiotics on the deconjugation of valproic acid glucuronide - Masuo_2010_Drug.Metab.Dispos_38_1828
Author(s) : Masuo Y , Ito K , Yamamoto T , Hisaka A , Honma M , Suzuki H
Ref : Drug Metabolism & Disposition: The Biological Fate of Chemicals , 38 :1828 , 2010
Abstract : Serum concentrations of valproic acid (VPA) are markedly decreased by coadministration of carbapenem antibiotics (CBPMs). Although inhibition of deconjugation of VPA-glucuronide (VPA-G) to VPA by CBPMs has been proposed as one of the mechanisms to account for this drug-drug interaction, little information is available on the mode of inhibition. In the present study, we characterized the enzyme involved in the deconjugation of VPA-G by using human and rat liver cytosol. It is suggested that 1) deconjugation activity inhibited by CBPMs may be selective for VPA-G, 2) deconjugation of VPA-G may be mediated by enzyme(s) other than beta-glucuronidase, and 3) the irreversible inactivation may be responsible for the inhibition of deconjugation of VPA-G by CBPMs. Finally, the kinetic parameters for inactivation (K'(app) and k(inact)) were determined for four CBPMs of diverse structure from in vitro experiments. Based on the results of simulation analyses with these parameters and the degradation rate constant of the putative VPA-G deconjugation enzyme obtained from experiments using rats, it is probable that the deconjugation enzyme for VPA-G in the liver is rapidly and mostly inactivated by these CBPMs under clinical situations.
ESTHER : Masuo_2010_Drug.Metab.Dispos_38_1828
PubMedSearch : Masuo_2010_Drug.Metab.Dispos_38_1828
PubMedID: 20581094

Title : Inhibition of acetylcholinesterase by metabolites of copper pyrithione (CuPT) and its possible involvement in vertebral deformity of a CuPT-exposed marine teleostean fish - Mochida_2009_Comp.Biochem.Physiol.C.Toxicol.Pharmacol_149_624
Author(s) : Mochida K , Ito K , Harino H , Tanaka H , Onduka T , Kakuno A , Fujii K
Ref : Comparative Biochemistry & Physiology C Toxicol Pharmacol , 149 :624 , 2009
Abstract : In a previous study, we demonstrated that exposure to an antifouling biocide, copper pyrithione (CuPT), early during life induced vertebral deformity in the larvae of a marine fish, the mummichog (Fundulus heteroclitus). Skeletal deformities may be caused by inhibition by of acetylcholiensterase (AChE) activity, and to elucidate the mechanism underlying the CuPT-associated vertebral deformity, we first examined whether CuPT, zinc pyrithione (ZnPT), and their degradation products could inhibit AChE activity in the fish. Two of the degradation products, 2,2'-dipyridyldisulfide [(PS)(2)] and 2,2'-dithiobispyridine-N-oxide [(PT)(2)], but neither CuPT nor ZnPT, exhibited prominent AChE-inhibiting activity. Secondly, thin-layer chromatography revealed that mummichog hepatic microsomes metabolized CuPT to produce (PS)(2) in a microsome-dependent manner. The AChE inhibition induced in CuPT-exposed fish is likely due to (PS)(2) that was produced through metabolism of acquired CuPT. (PS)(2) may cause therefore skeletal deformity in CuPT-exposed fish by means of its neuromuscular blocking properties, through a mechanism similar to that proposed for animals exposed to organophosphorous pesticides.
ESTHER : Mochida_2009_Comp.Biochem.Physiol.C.Toxicol.Pharmacol_149_624
PubMedSearch : Mochida_2009_Comp.Biochem.Physiol.C.Toxicol.Pharmacol_149_624
PubMedID: 19211040

Title : Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue - Nakajima_2008_J.Bacteriol_190_7819
Author(s) : Nakajima Y , Ito K , Toshima T , Egawa T , Zheng H , Oyama H , Wu YF , Takahashi E , Kyono K , Yoshimoto T
Ref : Journal of Bacteriology , 190 :7819 , 2008
Abstract : The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.
ESTHER : Nakajima_2008_J.Bacteriol_190_7819
PubMedSearch : Nakajima_2008_J.Bacteriol_190_7819
PubMedID: 18820015
Gene_locus related to this paper: xanma-P95782

Title : Early life-stage toxicity test for copper pyrithione and induction of skeletal anomaly in a teleost, the mummichog (Fundulus heteroclitus) - Mochida_2008_Environ.Toxicol.Chem_27_367
Author(s) : Mochida K , Ito K , Harino H , Onduka T , Kakuno A , Fujii K
Ref : Environ Toxicol Chem , 27 :367 , 2008
Abstract : We used a teleost fish, the mummichog (Fundulus heteroclitus), to conduct early life-stage toxicity testing for copper pyrithione (CuPT). Fertilized mummichog eggs were exposed to CuPT at various concentrations for 50 d under continuous flow-through conditions. Hatchability, survival, growth, and morphologic abnormalities were measured. Hatchability did not differ significantly between any experimental group and control groups. Survival and growth were significantly reduced at 50 d in the groups exposed to 2 or 4 microg/L CuPT. During the test, morphologic abnormalities, such as vertebral deformity and formation of inflammatory masses in the lateral muscles, occurred in fish exposed to CuPT. Light and electron microscopic studies indicated that muscle dysfunction played a role in the vertebral deformity and revealed that the inflammatory mass was composed mainly of macrophages and necrotic myocytes. We consider that macrophages infiltrated and phagocytized necrotic cells, thus forming the inflammatory mass. In addition, acetylcholinesterase activity was markedly decreased in the 2- and 4-microg/L exposure groups, suggesting the skeletal deformity was due to mechanisms similar to those proposed for organophosphorous pesticide exposure.
ESTHER : Mochida_2008_Environ.Toxicol.Chem_27_367
PubMedSearch : Mochida_2008_Environ.Toxicol.Chem_27_367
PubMedID: 18348625

Title : Novel inhibitor for prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis and details of substrate-recognition mechanism - Xu_2008_J.Mol.Biol_375_708
Author(s) : Xu Y , Nakajima Y , Ito K , Zheng H , Oyama H , Heiser U , Hoffmann T , Gartner UT , Demuth HU , Yoshimoto T
Ref : Journal of Molecular Biology , 375 :708 , 2008
Abstract : A new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with a K(i) value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 A resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction intermediate between the enzyme and substrate. We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected. Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild-type and formed a hydrogen bond with Gln203. The k(cat)/K(M) values of the E636A mutant toward the two substrates used were smaller than those of the wild-type by two orders of magnitude. The K(i) value of our inhibitor for the E636A mutant was 48.8 microM, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate.
ESTHER : Xu_2008_J.Mol.Biol_375_708
PubMedSearch : Xu_2008_J.Mol.Biol_375_708
PubMedID: 18042490
Gene_locus related to this paper: porgi-q7muw6

Title : Unusual extra space at the active site and high activity for acetylated hydroxyproline of prolyl aminopeptidase from Serratia marcescens - Nakajima_2006_J.Bacteriol_188_1599
Author(s) : Nakajima Y , Ito K , Sakata M , Xu Y , Nakashima K , Matsubara F , Hatakeyama S , Yoshimoto T
Ref : Journal of Bacteriology , 188 :1599 , 2006
Abstract : The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 angstroms resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-betaNA (4-acetyloxyproline beta-naphthylamide) was a better substrate than Pro-betaNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.
ESTHER : Nakajima_2006_J.Bacteriol_188_1599
PubMedSearch : Nakajima_2006_J.Bacteriol_188_1599
PubMedID: 16452443
Gene_locus related to this paper: serma-impep

Title : D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol alters cellular cholesterol homeostasis by modulating the endosome lipid domains - Makino_2006_Biochemistry_45_4530
Author(s) : Makino A , Ishii K , Murate M , Hayakawa T , Suzuki Y , Suzuki M , Ito K , Fujisawa T , Matsuo H , Ishitsuka R , Kobayashi T
Ref : Biochemistry , 45 :4530 , 2006
Abstract : D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is a frequently used inhibitor of glycosphingolipid biosynthesis. However, some interesting characteristics of D-PDMP cannot be explained by the inhibition of glycolipid synthesis alone. In the present study, we showed that d-PDMP inhibits the activation of lysosomal acid lipase by late endosome/lysosome specific lipid, bis(monoacylglycero)phosphate (also called as lysobisphosphatidic acid), through alteration of membrane structure of the lipid. When added to cultured fibroblasts, D-PDMP inhibits the degradation of low-density lipoprotein (LDL) and thus accumulates both cholesterol ester and free cholesterol in late endosomes/lysosomes. This accumulation results in the inhibition of LDL-derived cholesterol esterification and the decrease of cell surface cholesterol. We showed that D-PDMP alters cellular cholesterol homeostasis in a glycosphingolipid-independent manner using L-PDMP, a stereoisomer of D-PDMP, which does not inhibit glycosphingolipid synthesis, and mutant melanoma cell which is defective in glycolipid synthesis. Altering cholesterol homeostasis by D-PDMP explains the unique characteristics of sensitizing multidrug resistant cells by this drug.
ESTHER : Makino_2006_Biochemistry_45_4530
PubMedSearch : Makino_2006_Biochemistry_45_4530
PubMedID: 16584188

Title : Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis - Ito_2006_J.Mol.Biol_362_228
Author(s) : Ito K , Nakajima Y , Xu Y , Yamada N , Onohara Y , Ito T , Matsubara F , Kabashima T , Nakayama K , Yoshimoto T
Ref : Journal of Molecular Biology , 362 :228 , 2006
Abstract : The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.
ESTHER : Ito_2006_J.Mol.Biol_362_228
PubMedSearch : Ito_2006_J.Mol.Biol_362_228
PubMedID: 16914159
Gene_locus related to this paper: porgi-q7muw6

Title : Risk factors for hospital-acquired bacteremia - Yoshida_2005_Intern.Med_44_1157
Author(s) : Yoshida T , Tsushima K , Tsuchiya A , Nishikawa N , Shirahata K , Kaneko K , Ito K , Kawakami H , Nakagawa S , Suzuki T , Kubo K , Ikeda S
Ref : Intern Med , 44 :1157 , 2005
Abstract : OBJECTIVE: Bacteremia is one of the most serious health problems associated with high morbidity and mortality. The aim of this study was to identify risk factors for bacteremia in daily medical care to facilitate rapid and accurate clinical decisions about treatment. PATIENTS AND
METHODS: We studied 306 inpatients retrospectively. Age, peripheral neutrophil count, C-reactive protein (CRP), platelets, serum total cholesterol, total protein, albumin and cholinesterase were compared in patients with positive- and negative-blood cultures. The associations between blood culture positivity and glucose tolerance, bedridden state, presence of a central venous catheter (CVC) or urinary catheter were examined. On October 14, 2002, strategies for prevention of catheter-related infection were altered in our hospital. We studied the impact of these changes on the risk of bacteremia.
RESULTS: Sixty-seven patients had positive and 239 had negative blood cultures. Age, neutrophil, platelets, total protein, albumin, and cholinesterase were significantly different between the culture-positive patients and the culture-negative patients. Multivariate analysis showed albumin and platelets as independent predictors. The bedridden state and catheter-inserted states (central venous or urinary) conferred significantly higher positive blood culture rates. Multivariate analysis showed using urinary catheters and indwelling femoral CVCs as independent risk factors. There was no significant difference in the blood culture-positive rate before and after the change in prevention strategies; before the change, 6 of 9 catheter-inserted blood culture-positive cases yielded MRSA, while 4 of 12 cultures yielded Staphylococcus epidermidis after the change. CONCLUSION: Our study highlights the risk factors of bacteremia in vulnerable patients.
ESTHER : Yoshida_2005_Intern.Med_44_1157
PubMedSearch : Yoshida_2005_Intern.Med_44_1157
PubMedID: 16357453

Title : Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis - Nakajima_2005_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_61_1046
Author(s) : Nakajima Y , Ito K , Xu Y , Yamada N , Onohara Y , Ito T , Yoshimoto T
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 61 :1046 , 2005
Abstract : A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 149.4, c = 159.7 A. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a VM value of 3.14 A3 Da(-1). Diffraction data were collected to 2.1 A resolution using synchrotron radiation at the BL5 station of the Photon Factory.
ESTHER : Nakajima_2005_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_61_1046
PubMedSearch : Nakajima_2005_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_61_1046
PubMedID: 16511231
Gene_locus related to this paper: porgi-q7muw6

Title : Novel prolyl tri\/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning - Umezawa_2004_J.Biochem_136_293
Author(s) : Umezawa Y , Yokoyama K , Kikuchi Y , Date M , Ito K , Yoshimoto T , Matsui H
Ref : J Biochem , 136 :293 , 2004
Abstract : The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.
ESTHER : Umezawa_2004_J.Biochem_136_293
PubMedSearch : Umezawa_2004_J.Biochem_136_293
PubMedID: 15598885
Gene_locus related to this paper: strmb-tpap

Title : Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism - Ito_2004_Mol.Microbiol_52_399
Author(s) : Ito K , Tanaka T , Hatta R , Yamamoto M , Akimitsu K , Tsuge T
Ref : Molecular Microbiology , 52 :399 , 2004
Abstract : The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host-specific toxins and cause diseases on different plants. The strawberry pathotype produces host-specific AF-toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK-toxin. The strawberry pathotype produces two related molecular species, AF-toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT-1 from the strawberry pathotype that contains three genes involved in AF-toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT-1. AFTS1 encodes a protein with similarity to enzymes of the aldo-ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: Delta aftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF-toxin II, but not AF-toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.
ESTHER : Ito_2004_Mol.Microbiol_52_399
PubMedSearch : Ito_2004_Mol.Microbiol_52_399
PubMedID: 15066029
Gene_locus related to this paper: altal-aft8 , altal-amt4

Title : Novel inhibitor for prolyl aminopeptidase from Serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor - Inoue_2003_Arch.Biochem.Biophys_416_147
Author(s) : Inoue T , Ito K , Tozaka T , Hatakeyama S , Tanaka N , Nakamura KT , Yoshimoto T
Ref : Archives of Biochemistry & Biophysics , 416 :147 , 2003
Abstract : Prolyl aminopeptidase from Serratia marcescens hydrolyzed x-beta-naphthylamides (x=prolyl, alanyl, sarcosinyl, L-alpha-aminobutylyl, and norvalyl), which suggested that the enzyme has a pocket for a five-member ring. Based on the substrate specificity, novel inhibitors of Pro, Ala, and Sar having 2-tert-butyl-[1,3,4]oxadiazole (TBODA) were synthesized. The K(i) value of Pro-TBODA, Ala-TBODA, and Sar-TBODA was 0.5 microM, 1.6 microM, and 12mM, respectively. The crystal structure of enzyme-Pro-TBODA complex was determined. Pro-TBODA was located at the active site. Four electrostatic interactions were located between the enzyme and the amino group of Pro inhibitors (Glu204:0E1-N:Inh, Glu204:0E2-N:Inh, Glu232:0E1-N:Inh, and Gly46:O-N:Inh), and the residue of the inhibitors was inserted into the hydrophobic pocket composed of Phe139, Leu141, Leu146, Tyr149, Tyr150, and Phe236. The roles of Phe139, Tyr149, and Phe236 in the hydrophobic pocket and Glu204 and Glu232 in the electrostatic interactions were confirmed by site-directed mutagenesis, which indicated that the molecular recognition of proline is achieved through four electrostatic interactions and an insertion in the hydrophobic pocket of the enzyme.
ESTHER : Inoue_2003_Arch.Biochem.Biophys_416_147
PubMedSearch : Inoue_2003_Arch.Biochem.Biophys_416_147
PubMedID: 12893291
Gene_locus related to this paper: serma-impep

Title : A conditionally dispensable chromosome controls host-specific pathogenicity in the fungal plant pathogen Alternaria alternata - Hatta_2002_Genetics_161_59
Author(s) : Hatta R , Ito K , Hosaki Y , Tanaka T , Tanaka A , Yamamoto M , Akimitsu K , Tsuge T
Ref : Genetics , 161 :59 , 2002
Abstract : The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce host-specific toxins and cause diseases on different plants. Previously, the gene cluster involved in host-specific AK-toxin biosynthesis of the Japanese pear pathotype was isolated, and four genes, named AKT genes, were identified. The AKT homologs were also found in the strawberry and tangerine pathotypes, which produce AF-toxin and ACT-toxin, respectively. This result is consistent with the fact that the toxins of these pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety. In this study, three of the AKT homologs (AFT1-1, AFTR-1, and AFT3-1) were isolated on a single cosmid clone from strain NAF8 of the strawberry pathotype. In NAF8, all of the AKT homologs were present in multiple copies on a 1.05-Mb chromosome. Transformation-mediated targeting of AFT1-1 and AFT3-1 in NAF8 produced AF-toxin-minus, nonpathogenic mutants. All of the mutants lacked the 1.05-Mb chromosome encoding the AFT genes. This chromosome was not essential for saprophytic growth of this pathogen. Thus, we propose that a conditionally dispensable chromosome controls host-specific pathogenicity of this pathogen.
ESTHER : Hatta_2002_Genetics_161_59
PubMedSearch : Hatta_2002_Genetics_161_59
PubMedID: 12019223
Gene_locus related to this paper: altal-aft8

Title : Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens - Ito_2000_J.Biochem_128_673
Author(s) : Ito K , Inoue T , Kabashima T , Kanada N , Huang HS , Ma X , Azmi N , Azab E , Yoshimoto T
Ref : J Biochem , 128 :673 , 2000
Abstract : Molecular cloning of the gene and the crystal structure of the prolyl aminopeptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J. Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat)/K(m)), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.
ESTHER : Ito_2000_J.Biochem_128_673
PubMedSearch : Ito_2000_J.Biochem_128_673
PubMedID: 11011150
Gene_locus related to this paper: serma-impep

Title : Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli - Toida_2000_FEMS.Microbiol.Lett_189_159
Author(s) : Toida J , Fukuzawa M , Kobayashi G , Ito K , Sekiguchi J
Ref : FEMS Microbiology Letters , 189 :159 , 2000
Abstract : Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.
ESTHER : Toida_2000_FEMS.Microbiol.Lett_189_159
PubMedSearch : Toida_2000_FEMS.Microbiol.Lett_189_159
PubMedID: 10930731
Gene_locus related to this paper: aspor-TGLA

Title : Crystal structure of prolyl aminopeptidase from Serratia marcescens - Yoshimoto_1999_J.Biochem_126_559
Author(s) : Yoshimoto T , Kabashima T , Uchikawa K , Inoue T , Tanaka N , Nakamura KT , Tsuru M , Ito K
Ref : J Biochem , 126 :559 , 1999
Abstract : Prolyl aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.
ESTHER : Yoshimoto_1999_J.Biochem_126_559
PubMedSearch : Yoshimoto_1999_J.Biochem_126_559
PubMedID: 10467172
Gene_locus related to this paper: serma-impep

Title : The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease - Odagaki_1999_Structure.Fold.Des_7_399
Author(s) : Odagaki Y , Hayashi A , Okada K , Hirotsu K , Kabashima T , Ito K , Yoshimoto T , Tsuru D , Sato M , Clardy J
Ref : Structure Fold Des , 7 :399 , 1999
Abstract : BACKGROUND: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP. RESULTS: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81.
CONCLUSIONS: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.
ESTHER : Odagaki_1999_Structure.Fold.Des_7_399
PubMedSearch : Odagaki_1999_Structure.Fold.Des_7_399
PubMedID: 10196127

Title : Prolyl endopeptidase from Sphingomonas capsulata: isolation and characterization of the enzyme and nucleotide sequence of the gene - Kabashima_1998_Arch.Biochem.Biophys_358_141
Author(s) : Kabashima T , Fujii M , Meng Y , Ito K , Yoshimoto T
Ref : Archives of Biochemistry & Biophysics , 358 :141 , 1998
Abstract : Prolyl endopeptidase (prolyl oligopeptidase, EC 3.4.21.26) was purified from Sphingomonas capsulata IFO 12533, and its gene was cloned and expressed in Escherichia coli. The recombinant enzyme was markedly inhibited by diisopropyl phosphofluoridate and hardly affected by SH reagents or metal chelators, similar to the native enzyme purified from S. capsulata. Nucleotide sequencing analysis revealed an open reading frame of 2169 bp, coding for a protein of 723 amino acids with a predicted molecular weight of 78,433. The amino acid sequence was 39.6, 45.3, 38.9, and 38.3% homologous to Flavobacterium meningosepticum, Aeromonas hydrophila, porcine brain, and human T cell prolyl endopeptidase, respectively. A region near the C-terminus and the region containing the putative catalytic triad residues were highly conserved. The enzyme was crystallized by the hanging drop vapor diffusion method, using ammonium sulfate as a precipitant.
ESTHER : Kabashima_1998_Arch.Biochem.Biophys_358_141
PubMedSearch : Kabashima_1998_Arch.Biochem.Biophys_358_141
PubMedID: 9750174
Gene_locus related to this paper: sphca-Q9ZNM8

Title : Cloning and expression of the cDNA encoding prolyl oligopeptidase (prolyl endopeptidase) from bovine brain - Yoshimoto_1997_Biol.Pharm.Bull_20_1047
Author(s) : Yoshimoto T , Miyazaki K , Haraguchi N , Kitazono A , Kabashima T , Ito K
Ref : Biol Pharm Bull , 20 :1047 , 1997
Abstract : Prolyl oligopeptidase (EC 3.4.21.26, prolyl endopeptidase) cDNA from bovine brain was cloned by PCR, and the amplified fragment was used as a probe to screen the cDNA library from bovine brain. The obtained clone contained a 2.7 kb DNA fragment with an open reading frame of 2130 nucleotides, and encoded a protein of 710 amino acids with a deduced molecular weight of 80640 Da. The deduced amino acid sequence is 95, 94, 51 and 48% homologous to those of human T-cell, porcine brain, Aeromonas hydrophila, and Flavobacterium meningosepticum prolyl endopeptidases, respectively. The bovine brain prolyl endopeptidase-encoding cDNA was expressed using an expression vector bearing a tac promoter, with an approximate yield of 20 micrograms/ml of cell culture.
ESTHER : Yoshimoto_1997_Biol.Pharm.Bull_20_1047
PubMedSearch : Yoshimoto_1997_Biol.Pharm.Bull_20_1047
PubMedID: 9353562
Gene_locus related to this paper: bovin-ppce

Title : Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme - Kabashima_1997_J.Biochem_122_601
Author(s) : Kabashima T , Kitazono A , Kitano A , Ito K , Yoshimoto T
Ref : J Biochem , 122 :601 , 1997
Abstract : We cloned and sequenced the Serratia marcescens prolyl aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36,083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36,000 and 38,000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3,4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a= b =65.6 A, and c=169.8 A, a space group P4(1)2(1)2 or P4(3)2(1)2, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 A resolution.
ESTHER : Kabashima_1997_J.Biochem_122_601
PubMedSearch : Kabashima_1997_J.Biochem_122_601
PubMedID: 9348090
Gene_locus related to this paper: serma-impep

Title : Dipeptidyl peptidase IV from Xanthomonas maltophilia: sequencing and expression of the enzyme gene and characterization of the expressed enzyme - Kabashima_1996_J.Biochem_120_1111
Author(s) : Kabashima T , Ito K , Yoshimoto T
Ref : J Biochem , 120 :1111 , 1996
Abstract : The dipeptidyl peptidase IV [EC 3.4.14.5] gene of Xanthomonas maltophilia, expressed in Escherichia coli, was cloned by the shotgun method. Nucleotide sequence analysis revealed an open reading frame of 2,223 bp, coding for a protein of 741 amino acids with a predicted molecular weight of 82,080. The expressed enzyme was extracted with SDS, and the solubilized enzyme was purified about 1,030-fold on columns of Toyopearl HW65C, DEAE-Toyopearl twice, and hydroxyapatite, with an activity recovery of 50%. The enzyme hydrolyzed a proline-containing peptide at the penultimate position, and was inhibited by diisopropyl phosphofluoridate. The enzyme was most active at pH 8.5, and was stable between at pH 7.0 and 9.0. The molecular weight of the purified enzyme was estimated to be 83,000 and 165,000 by SDS-PAGE and gel filtration, respectively.
ESTHER : Kabashima_1996_J.Biochem_120_1111
PubMedSearch : Kabashima_1996_J.Biochem_120_1111
PubMedID: 9010758
Gene_locus related to this paper: xanma-P95782

Title : Prolyl aminopeptidase gene from Flavobacterium meningosepticum: cloning, purification of the expressed enzyme, and analysis of its sequence - Kitazono_1996_Arch.Biochem.Biophys_336_35
Author(s) : Kitazono A , Kabashima T , Huang HS , Ito K , Yoshimoto T
Ref : Archives of Biochemistry & Biophysics , 336 :35 , 1996
Abstract : In spite of the numerous studies regarding prolyl aminopeptidase, little is known about its mechanism and the significance of its similarity to a number of hydrolases of diverse specificity that belong to the alpha/beta hydrolase-fold family (Pseudomonas 2-hydroxymuconic semialdehyde hydrolase, atropinesterase, and 2-hydroxy-6-oxophenylhexa-2,4-dienoic acid hydrolase; human and rat epoxide hydrolases). We report the cloning and sequencing of the novel prolyl aminopeptidase gene from Flavobacterium meningosepticum (FPAP) which allowed a more comprehensive sequence comparison. FPAP was found to be a 35-kDa monomeric enzyme, releasing N-terminal proline but not hydroxyproline residues from small peptides and naphthylamide esters. Using the unweighted pair group method with arithmetic mean method, an evolutionary tree that depicts the probable relationship between the prolyl aminopeptidases and the alpha/beta hydrolase-fold enzymes was constructed. Since the alpha/beta hydrolase-fold family might also include the members of the prolyl oligopeptidase family (prolyl oligopeptidase, dipeptidyl peptidase IV, and prolyl carboxypeptidase), this proposal links all the known Pro-Y bond-cleaving proline-specific peptidases (prolyl oligopeptidase family, prolyl aminopeptidases, and prolinase) as enzymes with similar scaffolds and hydrolytic mechanisms. On the other hand, the enzymes that cleave X-Pro bonds are metalloenzymes grouped within the "pita-bread" fold family (aminopeptidase P and prolidase). Although the latter two enzymes show significant sequence homology, prolyl aminopeptidase, prolinase, and the members of the prolyl oligopeptidase family do not, and might share the alpha/beta hydrolase-fold scaffold. This rationale would explain the failure in finding a common "proline-recognizing motif" in the primary structures of these proline-specific peptidases.
ESTHER : Kitazono_1996_Arch.Biochem.Biophys_336_35
PubMedSearch : Kitazono_1996_Arch.Biochem.Biophys_336_35
PubMedID: 8951032
Gene_locus related to this paper: flame-pamp

Title : Prolyl aminopeptidase is also present in Enterobacteriaceae: cloning and sequencing of the Hafnia alvei enzyme-gene and characterization of the expressed enzyme - Kitazono_1996_J.Biochem_119_468
Author(s) : Kitazono A , Kitano A , Kabashima T , Ito K , Yoshimoto T
Ref : J Biochem , 119 :468 , 1996
Abstract : The Hafnia alvei prolyl aminopeptidase gene (hpap) was cloned and sequenced, the expressed enzyme (HPAP) was purified to homogeneity and thoroughly characterized. An open reading frame of 1,281 bp was found to code for the enzyme, resulting in a protein of 427 amino acids with a molecular weight of 48,577. HPAP resembles the Aeromonas sobria enzyme, having 45% identity and the same distinctive properties with respect to size and substrate specificities. Both enzyme show similar chromatographic behavior, and HPAP could be purified following the procedure previously described for the Aeromonas enzyme. HPAP was found to be resistant to diisopropylphosphofluoridate as are most of the prolyl aminopeptidases hitherto described. In spite of this similarity, no inhibition by 1 mM p-chloromercuribenzoate solution could be detected. Significant inhibition was, however, observed when the enzyme was incubated with 3,4-dichloroisocoumarin. This study confirms the presence of two types of prolyl aminopeptidases, of which the Hafnia and Aeromonas enzymes constitute one group and the Bacillus, Neisseria, and Lactobacillus enzymes the other, and describes the cloning of the first prolyl aminopeptidase gene from an Enterobacteriaceae.
ESTHER : Kitazono_1996_J.Biochem_119_468
PubMedSearch : Kitazono_1996_J.Biochem_119_468
PubMedID: 8830041
Gene_locus related to this paper: hafal-P94800

Title : Cloning, sequencing, and expression of the dipeptidyl peptidase IV gene from Flavobacterium meningosepticum in Escherichia coli - Kabashima_1995_Arch.Biochem.Biophys_320_123
Author(s) : Kabashima T , Yoshida T , Ito K , Yoshimoto T
Ref : Archives of Biochemistry & Biophysics , 320 :123 , 1995
Abstract : The dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) gene from Flavobacterium meningosepticum was cloned by Southern and colony hybridizations using probes amplified by PCR, and expressed in Escherichia coli DH1. E. coli DH1 harboring pFDP-H1, which was a subclone derived from the positive clone pFDP-1, showed 3.5-fold higher activity than F. meningosepticum. Nucleotide sequencing analysis revealed an open reading frame of 2133 bp, coding for a protein of 711 amino acids with a predicted molecular weight of 80,626. The expressed enzyme in E. coli DH1/pFDP-H1 was purified about 345-fold with an activity recovery of 12.3%. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160,000 by gel filtration, respectively, suggesting a dimeric form of the native enzyme. The deduced amino acid sequence of DP IV was homologous to those of the serine proteases of the "prolyl endopeptidase family." A sequence near the C-terminal region and the putative catalytic triad residues were well conserved among these enzymes.
ESTHER : Kabashima_1995_Arch.Biochem.Biophys_320_123
PubMedSearch : Kabashima_1995_Arch.Biochem.Biophys_320_123
PubMedID: 7793970
Gene_locus related to this paper: flame-Q47900

Title : Protease II from Moraxella lacunata: cloning, sequencing, and expression of the enzyme gene, and crystallization of the expressed enzyme - Yoshimoto_1995_J.Biochem_117_654
Author(s) : Yoshimoto T , Tabira J , Kabashima T , Inoue S , Ito K
Ref : Journal of Biochemistry , 117 :654 , 1995
Abstract : A gene coding for protease II (basic amino acid specific oligoendopeptidase) from Moraxella lacunata was cloned and expressed in Escherichia coli DH1. The transformant harboring a hybrid plasmid, pMPROII-12, with a 3.0-kbp insert at the PvuII-SacI site in pUC19, showed 23-fold higher enzyme activity than M. lacunata. The expressed enzyme from E. coli DH1/pMPROII-12 was purified by 40-80% ammonium sulfate fractionation, chromatography on DEAE-Toyopearl, and Sephadex G-150 gel filtration. The enzyme was most active at pH 6.5 and stable at pH 6.5-9.5. It had an optimum temperature of 35 degrees C for 5 min of reaction and was stable to up to 35 degrees C for 30 min at pH 7.0. Its molecular weight was estimated to be 80,000 by SDS-PAGE and gel-filtration analyses. It enzyme was inhibited by diisopropyl fluorophosphate (DFP) and classified as a serine endoprotease. Its amino acid sequence was 38% homologous to that of the E. coli protease II. By alignment with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-534, Asp-619, and His-654. The enzyme was crystallized by the hanging drop vapor diffusion method using PEG 4000 as precipitant.
ESTHER : Yoshimoto_1995_J.Biochem_117_654
PubMedSearch : Yoshimoto_1995_J.Biochem_117_654
PubMedID: 7629037
Gene_locus related to this paper: morla-ptrb

Title : Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis - Kitazono_1994_J.Biochem_116_943
Author(s) : Kitazono A , Ito K , Yoshimoto T
Ref : J Biochem , 116 :943 , 1994
Abstract : Prolyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations--the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.
ESTHER : Kitazono_1994_J.Biochem_116_943
PubMedSearch : Kitazono_1994_J.Biochem_116_943
PubMedID: 7896753
Gene_locus related to this paper: serma-impep

Title : Location of the protease II gene (ptrB) on the physical map of the Escherichia coli chromosome -
Author(s) : Kanatani A , Yoshimoto T , Nagai H , Ito K , Tsuru D
Ref : Journal of Bacteriology , 174 :7881 , 1992
PubMedID: 1447164
Gene_locus related to this paper: ecoli-ptrb