Publications by authors named "Masashi Takahashi"

313 Publications

Heat stress induces oxidative stress and activates the KEAP1-NFE2L2-ARE pathway in bovine endometrial epithelial cells.

Biol Reprod 2021 Jul 22. Epub 2021 Jul 22.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku Kita 9 Nishi 9, Sapporo 060-8589, Japan.

Heat stress adversely affects the reproductive function in cows. Although a relationship between heat stress and oxidative stress has been suggested, it has not been sufficiently verified in bovine endometrial epithelial cells. Here, we investigated whether oxidative stress is induced by heat stress in bovine endometrial epithelial cells under high temperature. Luciferase reporter assays showed that the reporter activity of heat shock element (HSE) and antioxidant responsive element (ARE) was increased in endometrial epithelial cells cultured under high temperature compared to that in cells cultured under basal (thermoneutral) temperature. Also, nuclear factor, erythroid 2 like 2 (NFE2L2), a master regulator of cellular environmental stress response, stabilized and the expression levels of antioxidant enzyme genes increased under high temperature. Immunostaining confirmed the nuclear localization of NFE2L2 in endometrial epithelial cells cultured under high temperature. Quantitative polymerase chain reaction analysis showed that the expression levels of representative inflammatory cytokine genes, such as prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin 8, were significantly decreased in endometrial epithelial cells cultured under high temperature compared to those in cells cultured under basal temperature. Thus, our results suggest that heat stress induces oxidative stress, whereas NFE2L2 plays a protective role in bovine endometrial epithelial cells cultured under heat stress conditions.
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http://dx.doi.org/10.1093/biolre/ioab143DOI Listing
July 2021

Loop-mediated isothermal amplification (LAMP) and machine learning application for early pregnancy detection using bovine vaginal mucosal membrane.

Biochem Biophys Res Commun 2021 Jul 9;569:179-186. Epub 2021 Jul 9.

Graduate School of Global Food Resources/Global Center for Food, Land and Water Resources, Hokkaido University, Sapporo, Hokkaido, 060-8589, Japan. Electronic address:

An early and accurate pregnancy diagnosis method is required to improve the reproductive performance of cows. Here we developed an easy pregnancy detection method using vaginal mucosal membrane (VMM) with application of Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) and machine learning. Cows underwent artificial insemination (AI) on day 0, followed by VMM-collection on day 17-18, and pregnancy diagnosis by ultrasonography on day 30. By RNA sequencing of VMM samples, three candidate genes for pregnancy markers (ISG15 and IFIT1: up-regulated, MUC16: down-regulated) were selected. Using these genes, we performed RT-LAMP and calculated the rise-up time (RUT), the first-time absorbance exceeded 0.05 in the reaction. We next determined the cutoff value and calculated accuracy, sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) for each marker evaluation. The IFIT1 scored the best performance at 92.5% sensitivity, but specificity was 77.5%, suggesting that it is difficult to eliminate false positives. We then developed a machine learning model trained with RUT of each marker combination to predict pregnancy. The model created with the RUT of IFIT1 and MUC16 combination showed high specificity (86.7%) and sensitivity (93.3%), which were higher compared to IFIT1 alone. In conclusion, using VMM with RT-LAMP and machine learning algorithm can be used for early pregnancy detection before the return of first estrus.
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http://dx.doi.org/10.1016/j.bbrc.2021.07.015DOI Listing
July 2021

Immunocytochemical evaluation of epithelial-mesenchymal transition in epithelial tumors of dogs and cats.

J Vet Med Sci 2021 Jul 8. Epub 2021 Jul 8.

Kagoshima University Veterinary Teaching Hospital, Joint Faculty of Veterinary Medicine, Kagoshima University.

Epithelial-mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescence-immunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n=22) and cats (n=9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti-vimentin antibodies. Enzymatic immunohistochemistry (IHC) revealed that 51.6% (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5% (5/8) of those cases. In cats, FICC could detect EMT in 100% (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.
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http://dx.doi.org/10.1292/jvms.21-0021DOI Listing
July 2021

Factors that predict ventricular arrhythmias in the late phase after acute myocardial infarction.

ESC Heart Fail 2021 Jun 25. Epub 2021 Jun 25.

Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba, Chuo-ku, 260-8677, Japan.

Aims: Little is known regarding factors that predict the occurrence of lethal ventricular arrhythmias (VAs) occurring after acute myocardial infarction (AMI). This observational cohort study aimed to identify factors that predicted lethal VAs during the late phase after AMI in patients with reduced left ventricular ejection fraction (LVEF).

Methods And Results: Data were collected from our AMI database regarding consecutive patients with an LVEF of ≤40% after AMI (January 2012 to July 2018). The 'late phase' was defined as ≥7 days after AMI onset, and the primary endpoint was defined as lethal VAs in the late phase. The study included 136 patients (82% men; mean age: 66 ± 13 years). The average LVEF at admission was 32.7 ± 8.2%. During a mean follow-up period of 20.7 months, 14 patients (10%) experienced lethal VAs, including ventricular fibrillation (n = 8) and sustained ventricular tachycardia (n = 10). Univariate analyses revealed that lethal VAs were predicted by age and LVEF at admission. Receiver operating characteristic curve analysis indicated that the optimal cut-off value was 23% for using the LVEF at admission to predict the primary endpoint (area under the curve: 0.77, P < 0.0001). Multivariable analysis also demonstrated that LVEF at admission was an independent predictor of the primary endpoint (risk ratio = 7.12, P = 0.001).

Conclusions: Lethal VAs in the late phase are common in patients with AMI, and reduced LVEF and cardiac function at admission play a significant role in the risk stratification for future lethal VAs in this population.
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http://dx.doi.org/10.1002/ehf2.13499DOI Listing
June 2021

The diagnosis of early pneumoconiosis in dust-exposed workers: comparison of chest radiography and computed tomography.

Acta Radiol 2021 Jun 7:2841851211022501. Epub 2021 Jun 7.

Department of Public Health & Nursing, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Background: Chest radiography (CR) is employed as the evaluation of pneumoconiosis; however, we sometimes encounter cases in which computed tomography (CT) is more effective in detecting subtle pathological changes or cases in which CR yields false-positive results.

Purpose: To compare CR to CT in the diagnosis of early-stage pneumoconiosis.

Material And Methods: CR and CT were performed for 132 workers with an occupational history of mining. We excluded 23 cases of arc-welder's lung. Five readers who were experienced chest radiologists or pulmonologists independently graded the pulmonary small opacities on CR of the remaining 109 cases. We then excluded 37 cases in which the CT data were not sufficient for grading. CT images of the remaining 72 cases were graded by the five readers. We also assessed the degree of pulmonary emphysema in those cases.

Results: The grade of profusion on CR (CR score) of all five readers was identical in only 5 of 109 cases (4.6%). The CR score coincided with that on CT in 40 of 72 cases (56%). The CT score was higher than that on CR in 13 cases (18%). On the other hand, the CT score was lower than that on CR in 19 cases (26%). The incidence of pulmonary emphysema was significantly higher in patients whose CR score was higher than their CT score.

Conclusion: CT is more sensitive than CR in the evaluation of early-stage pneumoconiosis. In cases with emphysema, the CR score tends to be higher in comparison to that on CT.
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http://dx.doi.org/10.1177/02841851211022501DOI Listing
June 2021

Requirement for expression of WW domain containing transcription regulator 1 in bovine trophectoderm development.

Biochem Biophys Res Commun 2021 05 1;555:140-146. Epub 2021 Apr 1.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

WW domain-containing transcription regulator 1 (WWTR1) is one of the primary effectors in the Hippo pathway, which plays essential roles in cell differentiation into trophectoderm (TE) and inner cell mass cell lineages at the blastocyst stage. However, little is known about the roles of WWTR1 in preimplantation development. The present study aimed to explore the significance of WWTR1 expression in preimplantation development using an mRNA knockdown (KD) system in bovine embryos. We first quantitated WWTR1 expression at protein and mRNA levels from fertilization to blastocyst stage. WWTR1 proteins gradually shifted from extranuclear localization during the 16-cell stage to nuclear localization by morula stage. WWTR1 mRNA expression was also transiently upregulated at the 16-cell stage. WWTR1 KD efficiently repressed WWTR1 expression at protein and mRNA levels. The WWTR1 KD embryos developed to the blastocyst stage at rates equivalent to those of controls, but TE cell numbers were significantly decreased. Representative TE-expressed genes, including CDX2 and IFNT were also significantly decreased in WWTR1 KD blastocysts. These results provide the first demonstration that WWTR1 expression is responsible for normal TE cell development in preimplantation embryos.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.112DOI Listing
May 2021

[Effects of Recommended Consultation Practice Using Fracture Risk Assessment Tool and Fracture Evaluation Questionnaire for Early Detection of Osteoporosis at Community Pharmacies].

Yakugaku Zasshi 2021 ;141(2):263-272

Department of Pharmaceutical and Health Care Management, Faculty of Pharmaceutical Sciences, Fukuoka University.

As one of the strategies for the early detection and treatment of osteoporosis, we have recommended visiting a hospital, based on the Fracture Risk Assessment Tool (FRAX) and evaluation questionnaire for osteoporotic fracture risk. In this study, we evaluated the impact of intervention by community pharmacists by integrating our data for the FRAX and questionnaire. The measurement of FRAX and the questionnaire survey were conducted through participation in health seminars organized by a community general support center from June 2018 to December 2019. Participants with a FRAX score more than 15% and at least one item in the questionnaire were considered to have "suspected osteoporosis" were recommended medical consultation. The medical treatment status for the participants considered to have "suspected osteoporosis" aged 40-90 years were analyzed. Of the 84 participants, 54 had a FRAX score more than 15%, and 44 participants fulfilled at least one item in the questionnaire. Medical consultation was recommended to 26 of these 44 participants, excluding 18 under treatment. Of the 25 participants, six (excluding one who disagreed) received consultation, and medical treatment was started for four of them (66.7%). However, consultation with the attending physician was recommended to five of the 18 participants who were initially on treatment but discontinued it at the time of the survey. Consequently, two participants resumed their osteoporosis treatment. Our data suggest advantages of community pharmacists' intervention using FRAX and a questionnaire for osteoporotic fracture risk measurement for early detection and medical treatment.
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http://dx.doi.org/10.1248/yakushi.20-00187DOI Listing
May 2021

Characteristics of interstitial lung disease in patients from post-marketing data on metastatic breast cancer patients who received abemaciclib in Japan.

Breast Cancer 2021 May 16;28(3):710-719. Epub 2021 Jan 16.

Kobe City Medical Center General Hospital, Kobe, Japan.

Background: This study evaluated characteristics of patients treated with abemaciclib and diagnosed with interstitial lung disease (ILD), using 12-month post-marketing data from the real-world setting in Japan.

Methods: Spontaneous reports of adverse events in patients receiving abemaciclib were collected regularly from healthcare providers (HCPs) from November 30, 2018, to November 29, 2019. Detailed follow-up was requested on suspected ILD cases via questionnaires and/or interviews. Radiological images (when available) were reviewed by an ILD adjudication committee of specialists. The age distribution of patients prescribed abemaciclib in Japan was estimated based on insurance claims data.

Results: Of 4700 patients estimated to be exposed to abemaciclib, 82 cases of ILD were reported (46 serious, 13 fatal). Most (91%) had ≥ 1 symptom at diagnosis, commonly dyspnea/shortness of breath (59%), cough (44%), and/or fever (37%). The majority (68%) received steroid therapy (24 [56%] recovered/recovering; 5 [12%] not recovered; 13 [30%] deaths, 1 [2.3%] unknown). No specific imaging patterns or sites of predilection were identified, but a diffuse alveolar damage (DAD) pattern was observed at outcome in 3 of 4 evaluated fatal cases (16 in total evaluated). Features of fatal cases included advanced age, pre-existing interstitial change, and advanced Eastern Cooperative Oncology Group Performance Status.

Conclusion: Advanced age and a DAD pattern were identified as potential risk factors for cases with poorer outcomes, as previously reported for drug-induced ILD. HCPs should consider the benefit-risk profile when prescribing abemaciclib, informing patients of risks and regularly monitoring treated patients to ensure early detection and treatment of ILD.
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http://dx.doi.org/10.1007/s12282-020-01207-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064939PMC
May 2021

Evaluation of Ductal Tissue in Coarctation of the Aorta Using X-Ray Phase-Contrast Tomography.

Pediatr Cardiol 2021 Mar 5;42(3):654-661. Epub 2021 Jan 5.

Department of Cardiovascular Surgery, Kobe Children's Hospital, 1-6-7, Minatojimaminamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.

We assessed the histological accuracy of X-ray phase-contrast tomography (XPCT) and investigated three-dimensional (3D) ductal tissue distribution in coarctation of the aorta (CoA) specimens. We used nine CoA samples, including the aortic isthmus, ductus arteriosus (DA), and their confluences. 3D images were obtained using XPCT. After scanning, the samples were histologically evaluated using elastica van Gieson (EVG) staining and transcription factor AP-2 beta (TFAP2B) immunostaining. XPCT sectional images clearly depicted ductal tissue distribution as low-density areas. In comparison with EVG staining, the mass density of the aortic wall positively correlated with elastic fiber formation (R = 0.69, P < 0.001). TFAP2B expression was consistent with low-density area including intimal thickness on XPCT images. On 3D imaging, the distances from the DA insertion to the distal terminal of the ductal media and to the intima on the ductal side were 1.63 ± 0.22 mm and 2.70 ± 0.55 mm, respectively. In the short-axis view, the posterior extension of the ductal tissue into the aortic lumen was 79 ± 18% of the diameter of the descending aorta. In three specimens, the aortic wall was entirely occupied by ductal tissue. The ductal intima spread more distally and laterally than the ductal media. The contrast resolution of XPCT images was comparable to that of histological assessment. Based on the 3D images, we conclude that complete resection of intimal thickness, including the opposite side of the DA insertion, is required to eliminate residual ductal tissue and to prevent postoperative re-coarctation.
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http://dx.doi.org/10.1007/s00246-020-02526-5DOI Listing
March 2021

Effect of summer heat stress on gene expression in bovine uterine endometrial tissues.

Anim Sci J 2020 Jan;91(1):e13474

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.

Heat stress negatively affects reproductive functions in cows. Increased temperature disturbs fetal development in utero. However, the effect of heat stress on uterine endometrial tissues has not been fully examined. Using qPCR analysis, we measured the mRNA expression of various molecular markers in uterine endometrial tissue of dairy cows from Hokkaido, Japan, in winter and summer. Markers examined were heat shock proteins (HSPs), antioxidant enzymes (catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase 4), inflammatory cytokines, and interferon stimulated genes. Our results showed heat stress, body and milk temperatures were higher during summer than during winter. Expression levels of HSP27, HSP60, and HSP90 mRNA, and of catalase and copper/zinc superoxide dismutase mRNA were lower in summer than in winter. Tumor necrosis factor alpha expression was higher in summer than in winter. In conclusion, summer heat stress may reduce the expression of HSPs, affecting the levels of inflammatory cytokines in bovine uterine endometrial tissue.
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http://dx.doi.org/10.1111/asj.13474DOI Listing
January 2020

Yes-associated protein 1 translocation through actin cytoskeleton organization in trophectoderm cells.

Dev Biol 2020 12 15;468(1-2):14-25. Epub 2020 Sep 15.

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku Kita 9 Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.
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http://dx.doi.org/10.1016/j.ydbio.2020.09.004DOI Listing
December 2020

Argyrophilic nucleolar organizer regions staining for cytology smears in dogs and cats.

J Vet Med Sci 2020 Sep 8;82(9):1267-1270. Epub 2020 Jul 8.

Kagoshima University Veterinary Teaching Hospital, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.

The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n=14) and cats (n=12). Acetone, formalin, ethanol and methanol were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May-Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.
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http://dx.doi.org/10.1292/jvms.20-0271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538313PMC
September 2020

Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes.

Mol Hum Reprod 2020 09;26(9):689-701

Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.
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http://dx.doi.org/10.1093/molehr/gaaa051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828578PMC
September 2020

The role of RHOA signaling in trophectoderm cell-fate decision in cattle.

Biochem Biophys Res Commun 2020 08 6;528(4):713-718. Epub 2020 Jun 6.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

Mammalian blastocysts are composed of two distinct cell lineages, namely the inner cell mass (ICM) and trophectoderm (TE). TE cells that give rise to the embryonic placenta are marked by an exclusive expression of the key determinant transcription factor, CDX2. Although Hippo signaling pathway is known to be responsible for this TE-specific expression of CDX2, the upstream regulator of this pathway in mammalian embryos is still controversial. In the present study, the involvement of the small molecular G protein, RHOA, in TE cell-fate decision in cattle was investigated. Inhibition of RHOA by the specific inhibitor, C3 transferase (C3), severely impaired the blastocyst formation. Further, C3 treatment significantly decreased the number of blastomeres with nuclearized YAP1, the prominent effector of Hippo pathway. An artificial isolation of ICM cells from blastocysts followed by the continuing culture to regenerate TE cells was conducted and showed that TE re-emergence from the isolated ICM is governed by Hippo pathway and suppressed by C3 treatment like that observed in developing embryos. Finally, the long-term exposure to C3 suggests the presence of alternative regulators of CDX2 expression other than RHOA signaling because there were still CDX2-positive cells after C3 treatment. These results demonstrated that RHOA signaling plays a significant role in TE cell-fate decision by regulating Hippo pathway in cattle.
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http://dx.doi.org/10.1016/j.bbrc.2020.05.210DOI Listing
August 2020

Reassessment of the Location of the Conduction System in Atrioventricular Septal Defect Using Phase-Contrast Computed Tomography.

Semin Thorac Cardiovasc Surg 2020 Winter;32(4):960-968. Epub 2020 May 23.

Division of Biomedical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

The location of the atrioventricular conduction axis in the setting of atrioventricular septal defect has previously been shown by histology and intraoperative recordings. We have now reassessed the arrangement using phase-contrast computed tomography, aiming to provide precise measurements so as to optimize future surgical repairs. We used the system based on an X-ray Talbot grating interferometer using the beamline BL20B2 in a SPring-8 synchrotron radiation facility available in Japan. We analyzed 18 specimens. The atrioventricular node was found within a nodal triangle 1.7 mm from the coronary sinus, with 95% confidence intervals from 1.45 to 2.0 millimeters. The depth of the node from the right atrial endocardium was 1.0 mm, with 95% confidence intervals from 0.73 to 1.34 mm. The overall length of the scooped-out ventricular septum was 30.8 mm, with 95% confidence intervals from 27.5 to 34.1 millimeters. The length from the inferior atrioventricular junction to the take-off of the right bundle branch was 12.8 mm, with 95% confidence intervals from 11.12 to 14.38 mm, giving a ratio of 0.43 for the extent of the axis along the inferior septum, with 95% confidence intervals of 0.38-0.48. The length of the non-branching bundle was 6.6 mm, with 95% confidence intervals from 5.57 to 7.7 mm. The proportion of septum occupied by the non-branching bundle was 0.22, with 95% confidence intervals from 0.18 to 0.26. Our findings confirm previous histological studies, extending them by providing precise measurements to guide placement of sutures during surgical repair.
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http://dx.doi.org/10.1053/j.semtcvs.2020.03.011DOI Listing
April 2021

Adverse impact of univentricular pacing for the patient with functional single ventricle: successful conversion to cardiac resynchronization therapy.

Surg Case Rep 2020 May 13;6(1):101. Epub 2020 May 13.

Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori, Niigata City, 951-8510, Japan.

Background: In a Fontan candidate, univentricular pacing may cause delay in interventricular conduction, which induces asynchronous contraction. Cardiac resynchronization therapy is expected to be an effective mode of therapy in such a case.

Case Presentation: A 7-month-old girl, diagnosed with dextrocardia, congenitally corrected transposition of the great artery [situs solitus, L-loop, and L-transposition], ventricular septal defect, infundibular and pulmonary valvular stenosis, and straddling of the tricuspid valve, was considered as a candidate for the Fontan procedure. She had undergone Blalock-Taussig shunt, and epicardial univentricular pacemaker implantation for persistent complete atrioventricular block. She underwent a bidirectional cavopulmonary shunt concomitant with ventricular lead translocation from the morphological left ventricle to the morphological right ventricle. After discharge, ventricular dyssynchrony was noted and cardiac failure persisted. She was converted to cardiac resynchronization therapy (CRT) at 13 months of age. Two-dimensional speckle tracking imaging was used by cardiologists to determine the most suitable pacing site. CRT rapidly corrected the heart failure; thus, she underwent the Fontan procedure after 1.5 years. Five years have passed since the cardiac resynchronization therapy; her interventricular synchrony is maintained well and the level of brain natriuretic peptide remains within normal range.

Conclusion: We describe the successful conversion from single ventricular pacing to CRT, in a case of congenitally corrected transposition of the great artery indicated for the Fontan procedure. The long-term prognosis of cardiac resynchronization therapy is undetermined in the pediatric population; therefore, further follow-up is required.
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http://dx.doi.org/10.1186/s40792-020-00863-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221050PMC
May 2020

Systemic Inonotus sp. Infection in a dog.

J Vet Med Sci 2020 Jun 4;82(6):779-783. Epub 2020 May 4.

Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.

A 3-years-old male golden retriever was presented for decreased activity (lethargy), anorexia, and titubation. Superficial lymph nodes were enlarged, and arrhythmia and tachycardia were auscultated. Fungal hyphae-like structures were detected in the biopsy samples from an enlarged lymph node and spleen. Nucleotide sequence of the internal transcribed spacer region of the fungi amplified by PCR was highly homologous to that of Inonotus pachyphloeus. The dog was treated with antifungal agents such as itraconazole, fluconazole, and voriconazole. Clinical signs resolved for 325 days but the dog died suddenly, possibly because of arrhythmia. Postmortem examination revealed the presence of a disseminated fungal infection. This report describes the case of canine systemic Inonotus sp. infection treated by an antifungal agent.
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http://dx.doi.org/10.1292/jvms.19-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324816PMC
June 2020

Effect of Long-term Administration of Prostaglandin E on Morphologic Changes in Ductus Arteriosus.

Ann Thorac Surg 2020 12 2;110(6):2088-2095. Epub 2020 Apr 2.

Department of Cardiovascular Surgery, Kobe Children's Hospital, Hyogo, Japan.

Background: To improve survival of patients with hypoplastic left heart syndrome, combination therapy with bilateral pulmonary artery banding and prostaglandin E (PGE)-mediated ductal patency was developed as an alternative for high-risk neonates in Japan. However, the effect of long-term PGE administration on ductus arteriosus remains unclear. Synchrotron radiation-based X-ray phase-contrast tomography (XPCT) enables clear visualization of soft tissues at an approximate spatial resolution of 12.5 μm. We aimed to investigate morphologic changes in ductus arteriosus after long-term PGE infusion using XPCT.

Methods: Seventeen ductus arteriosus tissue samples from patients with hypoplastic left heart syndrome were obtained during the Norwood procedure. The median duration of lipo-prostaglandin E (lipo-PGE) administration was 48 days (range, 3 to 123). Structural analysis of ductus arteriosus was performed and compared with conventional histologic analysis.

Results: The XPCT was successfully applied to quantitative measurements of ductal media. Significant correlation was found between the duration of lipo-PGE infusion and mass density of ductal media (R = 0.723, P = .001). The duration of lipo-PGE administration was positively correlated with elastic fiber staining (R = 0.799, P < .001) and negatively correlated with smooth muscle formation (R = -0.83, P < .001). No significant increase in intimal cushion formation was found after long-term lipo-PGE administration. Expression of ductus arteriosus dominant PGE-receptor EP4 almost disappeared in specimens when lipo-PGE was administered over 3 days.

Conclusions: Disorganized elastogenesis and little intimal cushion formation after long-term lipo-PGE administration suggest that ductus arteriosus remodeled to the elastic artery phenotype. Because EP4 was downregulated and ductus arteriosus exhibited elastic characteristics, the dosage of lipo-PGE might be decreased after a definite administration period.
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http://dx.doi.org/10.1016/j.athoracsur.2020.02.053DOI Listing
December 2020

Role of FOXO3A in Trastuzumab Combination Chemotherapy in Esophageal Squamous Cell Carcinoma.

Anticancer Res 2020 Apr;40(4):1921-1930

Department of Surgery, School of Medicine, Keio University, Tokyo, Japan.

Background/aim: Targeting of the human epidermal growth factor receptor 2 (HER2) is suggested to be beneficial for esophageal squamous cell carcinoma (ESCC) patients with HER2 amplification. In this study, we evaluated the effects of combination chemotherapy with HER2-targeted drug trastuzumab in ESCC cells and examined the underlying mechanism contributing to these effects.

Materials And Methods: HER2 expression was verified, and the efficacy of chemotherapy with and without trastuzumab was investigated in vitro and in vivo.

Results: The combination of trastuzumab and a combined-modality therapy stimulated the PI3K/Akt pathway in ESCC cells overexpressing HER2. Trastuzumab treatment resulted in the intranuclear accumulation of FOXO3A in ESCC xenografts overexpressing HER2. The combination of trastuzumab and a combined-modality therapy enhanced antitumor effects in HER2-overexpressing ESCC xenografts.

Conclusion: FOXO3A plays an important role in mediating the effects of trastuzumab, and combination chemotherapy may be a promising treatment for patients with HER2-overexpressing ESCC.
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http://dx.doi.org/10.21873/anticanres.14147DOI Listing
April 2020

Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

Reproduction 2020 06;159(6):757-766

Research Faculty of Agriculture, Hokkaido University, Hokkaido, Japan.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.
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http://dx.doi.org/10.1530/REP-20-0036DOI Listing
June 2020

Type-I interferon regulates matrix metalloproteinases clearance of the bovine endometrial spheroid.

Anim Sci J 2020 Jan-Dec;91(1):e13350

Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.

This study investigated the effect of type-I interferon (IFN) on the expression of matrix metalloproteinases (MMPs) of the bovine endometrial stromal cells (BES) and epithelial cells (BEE). The cells were separated and purified from the caruncles and cultured in DMEM/F-12 containing 10% fetal bovine serum. Spheroids were generated by using ascorbate. Zymograms of the supernatant showed that BEE predominantly expressed MMP-9, whereas MMP-2 was expressed in BES and homo-spheroids. While MMPs expression was not detected in hetero-spheroids. Real-time quantitative PCR revealed that type-I IFN and P4 suppressed the gene expression of MMP-2 and MMP-9 in hetero-spheroids, respectively. On the other hand, gelatin zymography analysis of the supernatant showed that type-I IFN strongly promote the clearance of MMPs. While zymograms of the MMPs stocked in the hetero-spheroids were significantly reduced by type-I IFN. Phenylmethanesulfonyl fluoride and leupeptin (both are serine proteinase inhibitors) significantly repressed the clearance of MMP-2 and MMP-9 induced by type-I IFN. Moreover, collagen fibers in hetero-spheroids significantly decreased after the treatment with type-I IFN. In conclusion, it was suggested that type-I IFN participate in the tissue remodeling by regulation the clearance of MMPs.
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http://dx.doi.org/10.1111/asj.13350DOI Listing
August 2020

Visualization and quantification of the atrioventricular conduction axis in hearts with ventricular septal defect using phase contrast computed tomography.

J Thorac Cardiovasc Surg 2020 Aug 19;160(2):490-496. Epub 2020 Feb 19.

Division of Biomedical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

Objective: To visualize and quantify the atrioventricular conduction axis in the setting of ventricular septal defect using phase contrast computed tomography.

Methods: We used the SPring-8 synchrotron radiation facility in Hyogo prefecture in Japan, processing and reconstructing the data with 3-dimensional software.

Results: We studied 8 hearts obtained from patients known to have had ventricular septal defects, aged from 6 to 150 days, with a median of 24.5 days. None of the individuals, however, had undergone corrective surgery. The penetrating bundle was found at a median of 1.43 mm from the septal crest, with a range of 0.99 to 1.54 mm. The distance to the nonbranching bundle to the right ventricular endocardium was 1.10 mm, with a range from 0.49 to 2.49 mm, to the origin of the left bundle branch was 2.46 mm, with a range from 1.7 to 3.18 mm, and to the origin of the right bundle branch was 2.34 mm, with a range from 0.50 to 2.59 mm. The median distance from the edge of the caudal limb of the septomarginal trabeculation to the right bundle branch was 1.04 mm, with a range from 0.81 to 1.16 mm.

Conclusions: We were able to show the precise location of the axis, with our findings suggesting that longitudinal sutures placed along the posteroinferior rim should be effective in avoiding iatrogenic injury, but sutures should not be placed in the valley between the limbs of the septomarginal trabeculation.
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http://dx.doi.org/10.1016/j.jtcvs.2020.02.036DOI Listing
August 2020

Overgrowth of mice generated from postovulatory-aged oocyte spindles.

FASEB Bioadv 2019 Jul 15;1(7):393-403. Epub 2019 Apr 15.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture Hokkaido University Sapporo Japan.

Oocyte spindle transfer (OST) is a potent reproductive technology used for mammals that enables the spindle in a deteriorated oocyte at the metaphase of the second meiotic division (MII) to serve as the genetic material for producing descendants. However, whether postnatal growth is achieved via OST using developmentally deteriorated MII oocytes remains unclear. At 16 h after human chorionic gonadotropin administration, denuded MII oocytes immediately after retrieval from oviducts (0 h-oocytes) were used for in vitro fertilization (IVF) as controls. For IVF using postovulatory-aged oocytes, the 0 h-oocytes were further incubated for 12 h and 24 h (12 h- and 24 h-oocytes). These mouse oocytes served as a model for assessing the postnatal growth of individuals produced via OST from developmentally deteriorated oocytes. The embryos from 12 h- and 24 h-oocyte spindles exhibited high rates of development up to the neonatal stage as good as the non-manipulated controls. However, the mice derived from the 24 h-oocyte spindles displayed heavier body weights and greater feed consumption than both controls and mice derived from 12 h-oocyte spindles. Our results demonstrate the feasibility of OST as a potent reproductive technology and its limitation in the use of excessively aged postovulatory oocytes in mammalian reproduction.
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http://dx.doi.org/10.1096/fba.2019-00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996386PMC
July 2019

A BODIPY-Based Fluorogenic Probe for Specific Imaging of Lipid Droplets.

Materials (Basel) 2020 Feb 3;13(3). Epub 2020 Feb 3.

Division of Materials Science, Graduate School of Environmental Science, Hokkaido University, Sapporo 0860-0810, Japan.

We developed an easily accessible boron-dipyrromethene (BODIPY)-based fluorogenic probe, which we named LD-TB. This probe emits bright fluorescence in oil; when compared with aqueous solution, a significant enhancement of fluorescence brightness is observed. Cellular experiments confirmed that the probe stains the lipid droplets (LDs) specifically in both live and fixed cells, providing background-free images. Compared with Nile Red dye, a commonly used LD marker, LD-TB showed superior photostability. The sharp absorption and emission bands enable its multicolor imaging with blue and green probes. Importantly, the probe has proved to have low toxicity and is compatible with cell fixation. Our research provides a promising new fluorogenic probe for specific imaging of LDs.
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http://dx.doi.org/10.3390/ma13030677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040634PMC
February 2020

Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes.

J Reprod Dev 2020 Feb 25;66(1):83-91. Epub 2019 Dec 25.

Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during invitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by invitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.
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http://dx.doi.org/10.1262/jrd.2019-123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040212PMC
February 2020

Evaluation of an automated point-of-care test system for measuring thrombin-antithrombin complex in dogs.

J Vet Emerg Crit Care (San Antonio) 2020 Jan 17;30(1):102-106. Epub 2019 Dec 17.

Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

Objective: To evaluate the utility of the chemiluminescent enzyme immunoassay (CLEIA) method for point-of-care (POC) measurement of canine plasma thrombin-antithrombin complex (TAT) concentration.

Assessment And Main Results: Plasma TAT concentration was measured in 54 healthy dogs and in 72 dogs with various diseases. A significant correlation was found between TAT concentration measured by CLEIA and that measured by an ELISA that was previously used in dogs. The upper limit of the reference value of TAT concentrations measured by CLEIA was determined to be 0.2 ng/mL based on the TAT concentration in 54 healthy dogs. TAT concentrations exceeded the reference interval in a portion of dogs when a hypercoagulable state may be present.

Conclusions: Canine plasma TAT concentrations measured using CLEIA were correlated with that measured using ELISA. Hence, a POC testing instrument may be used for early detection of activation of thrombin generation in emergency and critical care settings.
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http://dx.doi.org/10.1111/vec.12913DOI Listing
January 2020

[Evaluation of the Collaboration between Community Interprofessionals and Pharmacists for Early Detection of Dementia].

Yakugaku Zasshi 2019 ;139(12):1583-1590

Faculty of Pharmaceutical Sciences, Fukuoka University.

The purpose of this study is to evaluate the collaboration system between Muscat Pharmacy and community general support center by verifying the results of tests for early detection of dementia, and the early support response for the participants with suspected dementia by the center. From December 2016 to November 2018, we conducted measurement sessions using a touch panel-type test, MSP-1100 at 23 events that included health promotion classes and local community events directed by community general support center. At a later date, a visit survey was administered by the center to the participants with suspected dementia those who received a score lower than 12, and their individual records of early support responses and follow-ups were evaluated and analyzed. During the period, 597 participants had measurements taken with the touch panel-type test. Among them, 89 (14.9% of total) participants received a score lower than 12. The contents of the support by the center for the low-scoring participants were classified into nine categories. Forty of the 89 low-scoring participants were judged to have no problems. Others were cooperatively followed up with medical consultation recommendation, receiving home care management, and/or having their information provided physicians by the Muscat pharmacy and the center. The results of our study indicate that the cooperation between community pharmacy and community general support centers could allow for the early detection of dementia and follow-ups for suspected dementia.
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http://dx.doi.org/10.1248/yakushi.19-00098DOI Listing
February 2020

Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst.

J Biol Chem 2019 12 8;294(50):19209-19223. Epub 2019 Nov 8.

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo 060-8589, Japan

Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.
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http://dx.doi.org/10.1074/jbc.RA119.010746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916479PMC
December 2019

Structure-Based Design, Synthesis, and Biological Evaluation of Imidazo[4,5-b]Pyridin-2-one-Based p38 MAP Kinase Inhibitors: Part 2.

ChemMedChem 2019 12 7;14(24):2093-2101. Epub 2019 Nov 7.

Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.

We identified novel potent inhibitors of p38 mitogen-activated protein (MAP) kinase using a structure-based design strategy, beginning with lead compound, 3-(butan-2-yl)-6-(2,4-difluoroanilino)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (1). To enhance the inhibitory activity of 1 against production of tumor necrosis factor-α (TNF-α) in human whole blood (hWB) cell assays, we designed and synthesized hybrid compounds in which the imidazo[4,5-b]pyridin-2-one core was successfully linked with the p-methylbenzamide fragment. Among the compounds evaluated, 3-(3-tert-butyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)benzamide (25) exhibited potent p38 inhibition, superior suppression of TNF-α production in hWB cells, and also significant in vivo efficacy in a rat model of collagen-induced arthritis (CIA). In this paper, we report the discovery of potent, selective, and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.
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http://dx.doi.org/10.1002/cmdc.201900373DOI Listing
December 2019

Dynamic status of lysosomal cathepsin in bovine oocytes and preimplantation embryos.

J Reprod Dev 2020 Feb 3;66(1):9-17. Epub 2019 Nov 3.

Graduate School of Global Food Resources(GSF), Hokkaido University, Hokkaido 060-0809, Japan.

Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.
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http://dx.doi.org/10.1262/jrd.2019-115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040204PMC
February 2020
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