Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA(2) enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA(2). We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA(2) activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA(2)-lowering alleles. Results Lp-PLA(2) activity was decreased by 64% ( p = 2.4 x 10(-25)) with carriage of any of the four loss-of-function variants, by 45% ( p < 10(-300)) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 x 10(-12)) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA(2) activity by 65% ( p < 10(-300)). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA(2) activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA(2)-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA(2) is unlikely to be a causal risk factor.
Title: Micro-scale extraction and analysis of intact carboxylesterase after trapping on an immunoaffinity membrane surface Kimura A, Shimazaki Y Ref: Appl Biochem Biotechnol, 172:4053, 2014 : PubMed
Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm(2)) of this membrane and eluted by rinsing with 5 muL of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme's activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.
Title: Analysis of activity of esterase captured onto an immunoaffinity membrane Shimazaki Y, Sakikawa T, Kimura A Ref: Clinica Chimica Acta, 413:269, 2012 : PubMed
BACKGROUND: Specific proteins in biological fluids can be captured on an immunoaffinity membrane after polyclonal anti-porcine liver esterase antibodies are separated by non-denaturing 2-dimensional electrophoresis (2-DE) and transferred onto the membrane. The enzymatic activities of these captured proteins can then be monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). METHODS: Polyclonal anti-porcine liver esterase antibody was separated by non-denaturing 2-DE, transferred onto a polyvinylidene difluoride membrane and stained with Ponceau S. Esterase activity was examined by enzyme activity staining and MALDI-TOF MS after antigens, including purified carboxylesterase from porcine liver and cytosolic esterase from porcine retina, were captured on the immunoaffinity membrane. RESULTS: Esterase activity was detected on the immunoaffinity membrane after the enzyme was captured. Phosphatidylcholine hydrolysis by the esterase was monitored after the esterase was captured onto the membrane and attached to the target plate for MALDI-TOF MS. CONCLUSIONS: This method could be used to analyze changes in enzymatic activity under biological conditions such as health and disease conditions using immunoaffinity membranes and MALDI-TOF MS.
Title: [Acute massive gastrointestinal bleeding in the elderly] Kimura A, Iwamoto T Ref: Nihon Ronen Igakkai Zasshi, 46:250, 2009 : PubMed
AIM: Acute massive gastrointestinal bleeding appears to be becoming more frequent and complex in elderly patients with increasing longevity and a variety of diseases. because of long-term medication such as anti-thrombotic agents (ATA) for the prevention of cardiovascular diseases, anti-dementia drugs for the treatment of dementia, with increasing longevity and a variety of diseases. We, therefore, conducted a study to clarify these problems from a clinical view point in geriatric medicine. METHODS: The bleeding sites and the causes were studied in 85 consecutive patients with melena, hematemesis or acute blood loss, on the basis of clinical and emergency endoscopic findings. RESULTS: The patients were aged 66 to 95 (40 men and 45 women), and the underlying diseases were mainly cerebral infarction in the chronic phase, osteoarthropathy, atrial fibrillation, and dementia. Ten patients had a previous history of peptic ulcer. As initial symptoms, melena, hematemesis and acute anemia were seen in 49, 18 and 18 patients, respectively. Based on assessable endoscopic findings n=83), the bleeding sites were a gastroduodenal ulcer, reflux esophagitis/acute gastric mucosal lesion, colon diverticulum, or alimentary tract cancers in 43.4%, 13.2%, 16.9%, and 16.9%, respectively. A total of 75 patients were treated with some medications (on average 5.3 kinds of medication per patient). Non-steroidal anti-inflammatory drugs (NSAIDs) and/or ATA were common in 63.5% of 85 patients; particularly two thirds of the patients with hemorrhagic gastroduodenal ulcer had used non-aspirin NSAIDs for treatment of osteoarthropathy or acute upper respiratory inflammation, and/or low-dose aspirin for prevention of vascular events. Patients taking ATA over a long period had bleeding from various sites. Steroids, acetylcholinesterase inhibitor (AchEI), selective serotonin-reuptake inhibitor (SSRI), and bisphosphonates were taken by 5, 9, 3 and 3 patients, respectively, frequently in combination with NSAIDs or ATA. Cerebral infarction occurred in 3 of 38 patients after withdrawal of ATA. CONCLUSIONS: AchEI, SSRI and bisphosphonates, a newly developed group of drugs, have become widely available as geriatric medication. However, it appears that the incidence of drug-related gastrointestinal bleeding is extremely high not only in patients who underwent long-term treatment with NSAIDs or ATA, but also in patients treated chronically with AchEI, SSRI or bisphosphonates in combination with occasional use of NSAIDs. Therefore, to manage elderly patients safely, it is necessary to clarify both the drug and previous histories and consider NSAIDs use with caution even when they are indicated.
A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC(50), 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 nAChRs contribute to dopamine release from striatal minces; alpha-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that alpha-CtxArIB[V11L,V16D] selectively inhibits human and rat native alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.
Title: Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment Matsumiya Y, Wakita D, Kimura A, Sanpa S, Kubo M Ref: J Biosci Bioeng, 103:325, 2007 : PubMed
To construct an efficient lipid-containing wastewater treatment system, microorganisms that degrade lipids efficiently were isolated from various environmental sources. Strain DW2-1 showed the highest rate of degradation of 1% (w/v) salad oil among the isolated strains. Strain DW2-1 was identified as Burkholderia sp. and designated Burkholderia sp. DW2-1. The rate of degradation of salad oil, olive oil, sesame oil, and beef tallow by strain DW2-1 were 96.7%, 92.3%, 90.1% and 77.4%, respectively, during a 48-h cultivation. Strain DW2-1 grew well in a synthetic wastewater medium (>1 x 10(10) colony forming unit [CFU]/ml) between 20 degrees C and 38 degrees C, and its rate of degradation of salad oil was above 90% after a 48-h cultivation. The lipase and biosurfactant (BSF) activities of strain DW2-1 after a 48-h cultivation were 1720 U/l and 480 U/ml, respectively. In continuous cultures for lipid-containing wastewater treatment, DW2-1 was stably maintained and degraded more than 90% of salad oil during a 7-d cultivation.
Title: cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle Kimura A, Takahashi T Ref: Journal of Experimental Zoology, 286:656, 2000 : PubMed
A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids. The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases. The recombinant prolyl oligopeptidase generated in E. coli was purified and its properties were examined. The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme. Northern blot analysis showed wide expression of the prolyl oligopeptidase gene. Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase. These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression.
Title: Structure and localization of the mouse prolyl oligopeptidase gene Kimura A, Yoshida I, Takagi N, Takahashi T Ref: Journal of Biological Chemistry, 274:24047, 1999 : PubMed
We have cloned and characterized the genomic structure of the mouse gene for prolyl oligopeptidase that is mapped to chromosome 10B2-B3. The gene is about 92 kilobases in size and contains 15 exons. All exon-intron junction sequences conform to the GT/AG rule. Comparison with the presumed domain structures of the mouse prolyl oligopeptidase indicates that the propeller domain of the enzyme is encoded by exons 3-10, whereas the catalytic domain is encoded by exons 1-3 and 10-15. The catalytic triad residues are encoded by two exons (Ser(554) on exon 13 and His(680) and Asp(642) on exon 15). The 5'-flanking region of the mouse prolyl oligopeptidase gene has structural features found in housekeeping gene promoters, including a GC-rich segment and an absence of TATA and CAAT boxes. A primer extension assay showed the presence of multiple sites for the initiation of transcription. Transient transfection analysis demonstrated that the 5'-flanking region of the gene can direct efficient expression in COS1 cells. Deletion studies revealed that the downstream 125-base pair sequence of the region is required for promoter activity in the cells.
