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      • Enzymatic characterization of <i>in vitro</i>-expressed Baikal seal cytochrome P450 (CYP) 1A1, 1A2, and 1B1: Implication of low metabolic potential of CYP1A2 uniquely evolved in aquatic mammals

        Iwata, Hisato,Yamaguchi, Keisuke,Takeshita, Yoko,Kubota, Akira,Hirakawa, Shusaku,Isobe, Tomohiko,Hirano, Masashi,Kim, Eun-Young Elsevier 2015 Aquatic toxicology Vol.162 No.-

        <P><B>Abstract</B></P> <P>This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin <I>O</I>-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin <I>O</I>-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E<SUB>2</SUB>) were measured by using yeast-expressed Baikal seal (<I>Pusa sibirica</I>) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest <I>V</I> <SUB>max</SUB> of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. <I>V</I> <SUB>max</SUB>/<I>K</I> <SUB>m</SUB> ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>>PROD) similar to that of hCYP1B1; however, <I>V</I> <SUB>max</SUB>/<I>K</I> <SUB>m</SUB> ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E<SUB>2</SUB> to 2-OHE<SUB>2</SUB> and 4-OHE<SUB>2</SUB>, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E<SUB>2</SUB> by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the <I>V</I> <SUB>max</SUB>/<I>K</I> <SUB>m</SUB> ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E<SUB>2</SUB> hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16.2% and 83.7% of total CYP1s, respectively; bsCYP1B1 accounted for only 0.06%. Addition of anti-human CYP1A1 antibody in seal liver microsomes suppressed EROD activity more than did anti-human CYP1A2 antibody. Therefore, EROD may be catalyzed by hepatic bsCYP1A1 but not bsCYP1A2, consistent with the results of yeast-expressed bsCYP1A1 and 1A2. <I>In silico</I> substrate-docking models of bsCYP1s suggested that the defect in bsCYP1A2 enzymatic activities may be accounted for by the Pro substitution of highly conserved Thr in the I-helix, which is involved in formation of a hydrogen bond with the hydroperoxy intermediate on the heme. This Thr-Pro substitution is evolutionarily conserved across aquatic mammals and could explain their lower metabolic potential for persistent organic pollutants.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Catalytic activities of Baikal seal CYP1A1 were lower than those of human CYP1A1. </LI> <LI> Baikal seal CYP1B1 showed higher catalytic activities than human CYP1B1. </LI> <LI> Catalytic activities by Baikal seal CYP1A2 showed no or a minimal detectable value. </LI> <LI> Pro317 substitution appears to render seal CYP1A2 incapable of its catalytic function. </LI> <LI> This substitution is evolutionarily conserved in aquatic mammals. </LI> </UL> </P>

      • KCI등재

        Lymphopenia and Elevated Blood C-Reactive Protein Levels at Four Days Postoperatively Are Useful Markers for Early Detection of Surgical Site Infection Following Posterior Lumbar Instrumentation Surgery

        Eiichiro Iwata,Hideki Shigematsu,Munehisa Koizumi,Hiroshi Nakajima,Akinori Okuda,Yasuhiko Morimoto,Keisuke Masuda,Yasuhito Tanaka 대한척추외과학회 2016 Asian Spine Journal Vol.10 No.2

        Study Design: Case-control study. Purpose: To identify the characteristics of candidate indexes for early detection of surgical site infection (SSI). Overview of Literature: SSI is a serious complication of spinal instrumentation surgery. Early diagnosis and treatment are crucial for the welfare of the patient postoperation. Methods: We retrospectively reviewed laboratory data of patients who underwent posterior lumbar instrumentation surgery for degenerative spine disease. The sensitivity and specificity of six laboratory markers for early detection of SSI were calculated: greater elevation of the white blood cell count at day 7 than at day 4 postoperatively, greater elevation of the C-reactive protein (CRP) level at day 7 than at day 4 postoperatively, a CRP level of >10 mg/dL at 4 days postoperatively, neutrophil percentage of >75% at 4 days postoperatively, a lymphocyte percentage of <10% at 4 days postoperatively, and a lymphocyte count of <1,000/μL at 4 days postoperatively. Statistical analysis was via Fisher’s exact test and a p -value of <0.05 was considered significant. Results: In total, 85 patients were enrolled. Of these, five patients developed deep SSI. The sensitivity and specificity of each index were as follows: index 1, 20.0% and 77.5%; index 2, 20.0% and 83.8%; index 3, 40.0% and 97.5%; index 4, 40.0% and 86.3%; index 5, 0% and 96.3%; and index 6, 80.0% and 80.0%. A significant difference was noted for indexes 3 and 6. Conclusions: A CRP level of >10 mg/dL at 4 days postoperatively would be useful for definitive diagnosis of SSI, and a lymphocyte count of <1,000/μL at 4 days postoperatively would be a useful screening test for SSI. Although laboratory markers for early detection of SSI have been frequently reported, we believe that it is important to understand the characteristics of each index for a precise diagnosis.

      • KCI등재

        Lymphopenia at 4 Days Postoperatively Is the Most Significant Laboratory Marker for Early Detection of Surgical Site Infection Following Posterior Lumbar Instrumentation Surgery

        Eiichiro Iwata,Hideki Shigematsu,Akinori Okuda,Yasuhiko Morimoto,Keisuke Masuda,Hiroshi Nakajima,Munehisa Koizumi,Yasuhito Tanaka 대한척추외과학회 2016 Asian Spine Journal Vol.10 No.6

        Study Design: Case control study. Purpose: To identify the most significant laboratory marker for early detection of surgical site infection (SSI) using multiple logistic regression analysis. Overview of Literature: SSI is a serious complication of spinal instrumentation surgery. Early diagnosis and treatment are crucial. Methods: We retrospectively reviewed the laboratory data of patients who underwent posterior lumbar instrumentation surgery for degenerative spinal disease from January 2003 to December 2014. Six laboratory markers for early SSI detection were considered: renewed elevation of the white blood cell count, higher at 7 than 4 days postoperatively; renewed elevation of the C-reactive protein (CRP) level, higher at 7 than 4 days postoperatively; CRP level of >10 mg/dL at 4 days postoperatively; neutrophil percentage of >75% at 4 days postoperatively; lymphocyte percentage of <10% at 4 days postoperatively; and lymphocyte count of <1,000/μL at 4 days postoperatively. Results: Ninety patients were enrolled; five developed deep SSI. Multivariate regression analysis showed that a lymphocyte count of <1,000/μL at 4 days postoperatively was the sole significant independent laboratory marker for early detection of SSI (p =0.037; odds ratio, 11.9; 95% confidence interval, 1.2–122.7). Conclusions: A lymphocyte count of <1,000/μL at 4 days postoperatively is the most significant laboratory marker for early detection of SSI.

      • KCI등재

        Biceps-Related Physical Findings Are Useful to Prevent Misdiagnosis of Cervical Spondylotic Amyotrophy as a Rotator Cuff Tear

        Eiichiro Iwata,Hideki Shigematsu,Kazuya Inoue,Takuya Egawa,Masato Tanaka,Akinori Okuda,Yasuhiko Morimoto,Keisuke Masuda,Yusuke Yamamoto,Yoshihiro Sakamoto,Munehisa Koizumi,Yasuhito Tanaka 대한척추외과학회 2018 Asian Spine Journal Vol.12 No.1

        Study Design: Case–control study. Purpose: The aim of the present study was to identify physical findings useful for differentiating between cervical spondylotic amyotrophy (CSA) and rotator cuff tears to prevent the misdiagnosis of CSA as a rotator cuff tear. Overview of Literature: CSA and rotator cuff tears are often confused among patients presenting with difficulty in shoulder elevation. Methods: Twenty-five patients with CSA and 27 with rotator cuff tears were enrolled. We included five physical findings specific to CSA that were observed in both CSA and rotator cuff tear patients. The findings were as follows: (1) weakness of the deltoid muscle, (2) weakness of the biceps muscle, (3) atrophy of the deltoid muscle, (4) atrophy of the biceps muscle, and (5) swallow-tail sign (assessment of the posterior fibers of the deltoid). Results: Among 25 CSA patients, 10 (40.0%) were misdiagnosed with a rotator cuff tear on initial diagnosis. The sensitivity and specificity of each physical finding were as follows: (1) deltoid weakness (sensitivity, 92.0%; specificity, 55.6%), (2) biceps weakness (sensitivity, 80.0%; specificity, 100%), (3) deltoid atrophy (sensitivity, 96.0%; specificity, 77.8%), (4) biceps atrophy (sensitivity, 88.8%; specificity, 92.6%), and (5) swallow-tail sign (sensitivity, 56.0%; specificity, 74.1%). There were statistically significant differences in each physical finding. Conclusions: CSA is likely to be misdiagnosed as a rotator cuff tear; however, weakness and atrophy of the biceps are useful findings for differentiating between CSA and rotator cuff tears to prevent misdiagnosis.

      • KCI등재

        Reliability Comparison between “Distal Radius and Ulna” and “Simplified Tanner–Whitehouse III” Assessments for Patients with Adolescent Idiopathic Scoliosis

        Okuda Akinori,Shigematsu Hideki,Fujii Hiromasa,Iwata Eiichiro,Tanaka Masato,Morimoto Yasuhiko,Masuda Keisuke,Yamamoto Yusuke,Tanaka Yasuhito 대한척추외과학회 2020 Asian Spine Journal Vol.14 No.3

        Study Design: This is a retrospective clinical study.Purpose: In this study, we aim to evaluate the reliability of the distal radius and ulna assessment (DRU) and simplified Tanner-Whitehouse III classification (sTW3) in Japanese patients with adolescent idiopathic scoliosis (AIS).Overview of Literature: The greatest curvature of a scoliotic spine occurs at peak-height velocity (PHV), which is the time during which an individual’s height increases at the maximum rate. Diagnosing and appropriately treating AIS before PHV is the most effective way in order to prevent unnecessary deterioration of the scoliosis curve. Although it is difficult to detect scoliosis before PHV, DRU and sTW3, which involve evaluations using a left-hand radiograph, have been reported to be effective.Methods: We retrospectively evaluated 54 hands of 40 girls with AIS who visited Nara Medical University Hospital from 2000 to 2015 using previously collected radiographs. The examiners included a spine surgeon and a pediatric orthopedic surgeon, each with over 10 years of experience. The reliability of the DRU and sTW3 was evaluated using the kappa coefficient.Results: The left-hand radiographs of 40 female patients with AIS (mean age, 13.9±1.7 years; N=54 hands) were evaluated by two blinded examiners using the sTW3 and DRU methods. The highest inter-observer and intra-observer reliabilities (kappa, 0.64 and 0.62, respectively) for radius evaluation were determined. Radius evaluation by the DRU showed the highest agreement rate and smallest error between the inter- and intra-observer examinations.Conclusions: The DRU was the most reliable assessment tool, and it has the potential to be useful for precisely determining the stage of skeletal maturity in outpatient clinics.

      • KCI등재

        Recent topics on endoscopic ultrasonography-guided celiac plexus neurolysis

        Ichiro Yasuda,Tatsuyuki Hanaoka,Kosuke Takahashi,Yasuhiro Araki,Shinpei Doi,Takuji Iwashita,Keisuke Iwata,Tsuyoshi Mukai 소화기인터벤션의학회 2020 International journal of gastrointestinal interven Vol.9 No.4

        Endoscopic ultrasonography-guided celiac plexus neurolysis (EUS-CPN) is a widely practiced technique. Three sets of guidelines have recently been published and this procedure has become a major EUS technique. However, there are still several unanswered questions. The purpose of this manuscript is to review the recent literature pertaining to EUS-CPN. Currently, the main indication of EUS-CPN is pancreatic cancer pain. It is also performed for patients with chronic pancreatitis, but the indication is controversial due to its limited efficacy and a high incidence of infectious complications. Various techniques, such as central and bilateral EUS-CPN, and EUS-guided direct celiac ganglia neurolysis (EUS-CGN) have been performed. However, the efficacies of these techniques remain controversial. Complications related to the procedures are generally not serious, but major adverse events, such as paraplegia and ischemic complications, have been reported. The impacts of EUS-CPN on survival have also been evaluated. Although increased survival was expected via improvements in the quality of life, data suggests that EUS-CPN related procedures, especially EUS-CGN, might reduce the survival time. However, precise mechanisms have not been elucidated. In addition to conventional techniques, new techniques, such as EUS-guided celiac ganglion radiofrequency ablation (EUS-RFA) and the use of highly viscous phenol-glycerol, dexmedetomidine, and contrast-enhanced agents, have been introduced. However, these techniques are still in experimental stages. Additional studies need to be conducted to address these gaps in the literature.

      • KCI등재

        Recent topics on endoscopic ultrasonography-guided celiac plexus neurolysis

        Ichiro Yasuda,Tatsuyuki Hanaoka,Kosuke Takahashi,Yasuhiro Araki,Shinpei Doi,Takuji Iwashita,Keisuke Iwata,Tsuyoshi Mukai 소화기인터벤션의학회 2020 Gastrointestinal Intervention Vol.9 No.4

        Endoscopic ultrasonography-guided celiac plexus neurolysis (EUS-CPN) is a widely practiced technique. Three sets of guidelines have recently been published and this procedure has become a major EUS technique. However, there are still several unanswered questions. The purpose of this manuscript is to review the recent literature pertaining to EUS-CPN. Currently, the main indication of EUS-CPN is pancreatic cancer pain. It is also performed for patients with chronic pancreatitis, but the indication is controversial due to its limited efficacy and a high incidence of infectious complications. Various techniques, such as central and bilateral EUS-CPN, and EUS-guided direct celiac ganglia neurolysis (EUS-CGN) have been performed. However, the efficacies of these techniques remain controversial. Complications related to the procedures are generally not serious, but major adverse events, such as paraplegia and ischemic complications, have been reported. The impacts of EUS-CPN on survival have also been evaluated. Although increased survival was expected via improvements in the quality of life, data suggests that EUS-CPN related procedures, especially EUS-CGN, might reduce the survival time. However, precise mechanisms have not been elucidated. In addition to conventional techniques, new techniques, such as EUS-guided celiac ganglion radiofrequency ablation (EUS-RFA) and the use of highly viscous phenol-glycerol, dexmedetomidine, and contrast-enhanced agents, have been introduced. However, these techniques are still in experimental stages. Additional studies need to be conducted to address these gaps in the literature.

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