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Article ; Online: Establishment of a new method to isolate viable x-ray-sensitive cells from planarian by fluorescence-activated cell sorting.

Ishida, Miyuki / Kuroki, Yoshihito / Agata, Kiyokazu

2023 Volume 65, Issue 9, Page(s) 577–590

Abstract: Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian ... ...

Abstract	Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence-activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into x-ray-irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts, which can proliferate in the x-ray-irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes such as Hoechst 33342, DRAQ5, and DyeCycle, showed, more or less, toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the x-ray-sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% → ca. 89%), the sorted cells could actively proliferate when they were injected into x-ray-irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes.
MeSH term(s)	Animals ; Planarians ; Flow Cytometry/methods ; X-Rays ; Fluorescent Dyes/pharmacology ; Mammals
Chemical Substances	calcein AM (148504-34-1) ; Fluorescent Dyes
Language	English
Publishing date	2023-09-05
Publishing country	Japan
Document type	Journal Article
ZDB-ID	280433-5
ISSN	1440-169X ; 0012-1592
ISSN (online)	1440-169X
ISSN	0012-1592
DOI	10.1111/dgd.12886
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Article ; Online: Aspergillus oryzae α-l-rhamnosidase: Crystal structure and insight into the substrate specificity.

Makabe, Koki / Ishida, Naoki / Kanezaki, Nanako / Shiono, Yoshihito / Koseki, Takuya

Proteins

2023 Volume 92, Issue 2, Page(s) 236–245

Abstract: The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher ... ...

Abstract	The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to β-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to β-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that K
MeSH term(s)	Hesperidin ; Aspergillus oryzae/genetics ; Aspergillus oryzae/metabolism ; Substrate Specificity ; Kinetics ; Asparagine ; Glycoside Hydrolases/chemistry ; Rutin ; Glucosides
Chemical Substances	alpha-L-rhamnosidase (EC 3.2.1.40) ; Hesperidin (E750O06Y6O) ; Asparagine (7006-34-0) ; Glycoside Hydrolases (EC 3.2.1.-) ; Rutin (5G06TVY3R7) ; Glucosides
Language	English
Publishing date	2023-10-11
Publishing country	United States
Document type	Journal Article
ZDB-ID	806683-8
ISSN	1097-0134 ; 0887-3585
ISSN (online)	1097-0134
ISSN	0887-3585
DOI	10.1002/prot.26608
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Article ; Online: Mutational analysis of the effects of N-glycosylation sites on the activity and thermal stability of rutinosidase from Aspergillus oryzae.

Koseki, Takuya / Ishida, Naoki / Hirota, Ruka / Shiono, Yoshihito / Makabe, Koki

Enzyme and microbial technology

2022 Volume 161, Page(s) 110112

Abstract: Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-β-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the ... ...

Abstract	Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-β-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein. Among five single variants constructed for these sites, the N128D, N176D, and N359D variants exhibited similar mobility bands compared to that of the wild-type enzyme based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the N32D and N288D variants exhibited slightly and considerably increasing mobility bands, respectively. The N128D and N288D variants showed increasing and decreasing rutinosidase activity, respectively, compared to the case for the wild-type, without and with endo-H treatments. While the N128D and N176D variants had lower K
MeSH term(s)	Aspergillus oryzae/genetics ; Glycosylation ; Mutagenesis, Site-Directed ; Recombinant Proteins/metabolism
Chemical Substances	Recombinant Proteins
Language	English
Publishing date	2022-08-08
Publishing country	United States
Document type	Journal Article
ZDB-ID	423729-8
ISSN	1879-0909 ; 0141-0229
ISSN (online)	1879-0909
ISSN	0141-0229
DOI	10.1016/j.enzmictec.2022.110112
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Article ; Online: The effects of the first versus second glaucoma drainage implant surgery in patients with primary open-angle glaucoma.

Morita, Shuu / Hamanaka, Teruhiko / Sakurai, Tetsuro / Watanabe, Satoshi / Sakanishi, Yoshihito / Ishida, Nobuo / Ebihara, Nobuyuki

BMC ophthalmology

2023 Volume 23, Issue 1, Page(s) 509

Abstract: Background: To investigate the outcome of non-valved glaucoma drainage implant surgery (GDIS) in primary open-angle glaucoma (POAG) patients divided in the first GDI group (patients who underwent the first GDIS) and the second GDI group (patients who ... ...

Abstract	Background: To investigate the outcome of non-valved glaucoma drainage implant surgery (GDIS) in primary open-angle glaucoma (POAG) patients divided in the first GDI group (patients who underwent the first GDIS) and the second GDI group (patients who underwent the second GDIS because of the failed first GDIS). Methods: Intraocular pressure (IOP), visual acuity (VA), visual field defect (VFD), medication score (MS), survival rate of GDIS, complications, and patient background was retrospectively analyzed. Two success criteria were set: Criteria (1) IOP reduction ≥ 20% and 5 < IOP ≤ 21, Criteria (2) IOP reduction ≥ 20% and 5 < IOP ≤ 14. Results: There were 136 eyes of 109 patients in the first GDI group and 32 eyes of 27 patients in the second GDI group. In the first GDI group and II, mean preoperative IOP was 26.7 ± 6.7 mmHg and 23.7 ± 3.5 mmHg, respectively (P = 0.09). No statistically significant difference in postoperative IOP reduction was found between the two groups (P = 0.39). At 5-years postoperative, the Criteria 1 (Criteria 2) survival rate in the first GDI group and the second GDI group was 60.4% (31.7%) and 61.2% (25.6%), respectively (Criteria 1: hazard ratio [HR]: 0.64, 95% confidence interval [CI]: 0.30-1.35 [P = 0.24]; Criteria 2: HR: 0.81, 95% CI: 0.46-1.44, P = 0.48). No significant difference in VA, VFD change, MS, or complications was observed. Young patient age was the only significant factor for failure in the first GDI group (odds ratio: 0.95, 95% confidence interval: 0.91-1.00, P = 0.03). Conclusion: The second GDIS may be as effective as the first GDIS for IOP reduction in POAG patients, however, there is a high risk of failure in young-age patients and the surgery may be ineffective in eyes requiring Criteria 2.
MeSH term(s)	Humans ; Glaucoma/surgery ; Glaucoma, Open-Angle/surgery ; Glaucoma, Open-Angle/etiology ; Retrospective Studies ; Glaucoma Drainage Implants/adverse effects ; Intraocular Pressure ; Trabeculectomy ; Ocular Hypotension/etiology ; Guanine Nucleotide Dissociation Inhibitors ; Treatment Outcome
Chemical Substances	Guanine Nucleotide Dissociation Inhibitors
Language	English
Publishing date	2023-12-14
Publishing country	England
Document type	Journal Article
ZDB-ID	2050436-6
ISSN	1471-2415 ; 1471-2415
ISSN (online)	1471-2415
ISSN	1471-2415
DOI	10.1186/s12886-023-03247-y
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Article ; Online: Secretory co-factors in next-generation cellular therapies for cancer.

Okuma, Atsushi / Ishida, Yoshihito / Kawara, Taketo / Hisada, Shoji / Araki, Shinsuke

Frontiers in immunology

2022 Volume 13, Page(s) 907022

Abstract: Since chimeric antigen receptor (CAR) T-cell therapies for hematologic malignancies were approved by the U.S. Food and Drug Administration, numerous "next-generation" CAR T cells have been developed to improve their safety, efficacy, and applicability. ... ...

Abstract	Since chimeric antigen receptor (CAR) T-cell therapies for hematologic malignancies were approved by the U.S. Food and Drug Administration, numerous "next-generation" CAR T cells have been developed to improve their safety, efficacy, and applicability. Although some of these novel therapeutic strategies are promising, it remains difficult to apply these therapies to solid tumors and to control adverse effects, such as cytokine release syndrome and neurotoxicity. CAR T cells are generated using highly scalable genetic engineering techniques. One of the major strategies for producing next-generation CAR T cells involves the integration of useful co-factor(s) into the artificial genetic design of the CAR gene, resulting in next-generation CAR T cells that express both CAR and the co-factor(s). Many soluble co-factors have been reported for CAR T cells and their therapeutic effects and toxicity have been tested by systemic injection; therefore, CAR T cells harnessing secretory co-factors could be close to clinical application. Here, we review the various secretory co-factors that have been reported to improve the therapeutic efficacy of CAR T cells and ameliorate adverse events. In addition, we discuss the different co-factor expression systems that have been used to optimize their beneficial effects. Altogether, we demonstrate that combining CAR T cells with secretory co-factors will lead to next-generation CAR T-cell therapies that can be used against broader types of cancers and might provide advanced tools for more complicated synthetic immunotherapies.
MeSH term(s)	Cell- and Tissue-Based Therapy ; Cytokine Release Syndrome ; Humans ; Immunotherapy, Adoptive/methods ; Neoplasms ; Receptors, Chimeric Antigen ; United States
Chemical Substances	Receptors, Chimeric Antigen
Language	English
Publishing date	2022-08-18
Publishing country	Switzerland
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2022.907022
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Article: Development of novel clubroot resistant rapeseed lines (

Kawasaki, Mitsuyo / Ohara, Takayoshi / Ishida, Masahiko / Takahata, Yoshihito / Hatakeyama, Katsunori

Breeding science

2021 Volume 71, Issue 5, Page(s) 528–537

Abstract: Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from ... ...

Abstract	Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from rapeseed fields and found they were classified as pathotype groups 2 and 4 using Japanese F
Language	English
Publishing date	2021-10-12
Publishing country	Japan
Document type	Journal Article
ZDB-ID	1190370-3
ISSN	1344-7610 ; 0536-3683
ISSN	1344-7610 ; 0536-3683
DOI	10.1270/jsbbs.21014
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Article ; Online: Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts.

Minowa, Erika / Hayashi, Yoshinobu / Goh, Kenji / Ishida, Narumi / Kurashige, Yoshihito / Nezu, Akihiro / Saitoh, Masato / Tanimura, Akihiko

Journal of periodontal research

2023 Volume 58, Issue 2, Page(s) 274–282

Abstract: Background and objectives: Gingival overgrowth caused by phenytoin is proposed to be associated with Ca: Methods: Effects of 100 μM phenytoin on [Ca: Results: Exposure of HGFs to 100 μM phenytoin induced a transient increase in [Ca: Conclusion: ...

Abstract	Background and objectives: Gingival overgrowth caused by phenytoin is proposed to be associated with Ca Methods: Effects of 100 μM phenytoin on [Ca Results: Exposure of HGFs to 100 μM phenytoin induced a transient increase in [Ca Conclusion: Our findings demonstrate that phenytoin induced increase in [Ca
MeSH term(s)	Humans ; Phenytoin/adverse effects ; Gingiva ; Calcium ; Histamine/adverse effects ; Gingival Overgrowth/chemically induced ; Fibroblasts ; Adenosine Triphosphate/pharmacology ; Cells, Cultured
Chemical Substances	Phenytoin (6158TKW0C5) ; Calcium (SY7Q814VUP) ; Histamine (820484N8I3) ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2023-01-04
Publishing country	United States
Document type	Journal Article
ZDB-ID	390953-0
ISSN	1600-0765 ; 0022-3484
ISSN (online)	1600-0765
ISSN	0022-3484
DOI	10.1111/jre.13089
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Zs.A 599: Show issues

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Article ; Online: Quantitative Analytical Method for Single Rain Droplets via Crystal Formation in Photocrosslinking Polymer Gel.

Nanzai, Ben / Goto, Yoshiaki / Ishida, Yoshihito / Igawa, Manabu

Analytical sciences : the international journal of the Japan Society for Analytical Chemistry

2019 Volume 35, Issue 11, Page(s) 1263–1267

Abstract: Ion composition contained in individual rain droplets provides important information to investigate the chemistry in rain and clouds, but general rain sampling equipment temporally and spatially averages the information. Determination of the ... ...

Abstract	Ion composition contained in individual rain droplets provides important information to investigate the chemistry in rain and clouds, but general rain sampling equipment temporally and spatially averages the information. Determination of the SO
Language	English
Publishing date	2019-08-02
Publishing country	Japan
Document type	Journal Article
ZDB-ID	1483376-1
ISSN	1348-2246 ; 0910-6340
ISSN (online)	1348-2246
ISSN	0910-6340
DOI	10.2116/analsci.19P265
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Article: Mutational analysis of the effects of N-glycosylation sites on the activity and thermal stability of rutinosidase from Aspergillus oryzae

Koseki, Takuya / Ishida, Naoki / Hirota, Ruka / Shiono, Yoshihito / Makabe, Koki

Enzyme and microbial technology. 2022 Nov., v. 161

2022

Abstract	Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-β-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein. Among five single variants constructed for these sites, the N128D, N176D, and N359D variants exhibited similar mobility bands compared to that of the wild-type enzyme based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the N32D and N288D variants exhibited slightly and considerably increasing mobility bands, respectively. The N128D and N288D variants showed increasing and decreasing rutinosidase activity, respectively, compared to the case for the wild-type, without and with endo-H treatments. While the N128D and N176D variants had lower Kₘ values, the N288D and N359D variants had higher Kₘ values, compared to the wild-type, without and with endo-H treatments. Surprisingly, the N32D and N176D variants exhibited considerably greater thermal stability than the wild-type, without or with the endo-H treatments, whereas the N128D and N359D variants exhibited drastically decreased thermal stability. Circular dichroism (CD) spectra of the N128D and N359D variants showed a similar CD profile to that of the wild-type treated with endo-H; however, the molar ellipticity values of the peaks at 208 nm and 212 nm in the above variants varied from those of the intact wild-type and other variants.
Keywords	Aspergillus oryzae ; Komagataella pastoris ; circular dichroism spectroscopy ; crystal structure ; enzymes ; glycosylation ; mutational analysis ; polyacrylamide gel electrophoresis ; site-directed mutagenesis ; technology ; thermal stability
Language	English
Dates of publication	2022-11
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	423729-8
ISSN	1879-0909 ; 0141-0229
ISSN (online)	1879-0909
ISSN	0141-0229
DOI	10.1016/j.enzmictec.2022.110112
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Thin bronchoscopic cryobiopsy using a nasobronchial tube.

Oki, Masahide / Saka, Hideo / Kogure, Yoshihito / Niwa, Hideyuki / Ishida, Akane / Yamada, Arisa / Torii, Atsushi / Kitagawa, Chiyoe

BMC pulmonary medicine

2022 Volume 22, Issue 1, Page(s) 361

Abstract: Background: Transbronchial lung cryobiopsy is useful when diagnosing lung lesions. However, prevention of associated bleeding complications is essential. This study aimed to evaluate the safety and efficacy of our novel bronchoscopic cryobiopsy ... ...

Abstract	Background: Transbronchial lung cryobiopsy is useful when diagnosing lung lesions. However, prevention of associated bleeding complications is essential. This study aimed to evaluate the safety and efficacy of our novel bronchoscopic cryobiopsy technique, which uses a long nasobronchial tube to prevent blood flooding the central airway. Methods: Patients with localized or diffuse lung lesions were prospectively enrolled and underwent cryobiopsy using a 1.9 mm diameter cryoprobe and a 4.0 mm diameter thin bronchoscope under conscious sedation. For cryobiopsy, a long silicone tube (inner diameter, 5.0 mm) was advanced through the nose to the target bronchus, then wedged to drain blood under thin-tube bronchoscopic control. The primary endpoint was the frequency of bleeding complications. Results: Of the 80 patients initially enrolled, 73 that underwent at least one cryobiopsy were ultimately included. Mild bleeding during cryobiopsy occurred in 58 patients (79.5%), but there was no moderate or severe bleeding. Other complications occurred in four patients (two pneumothorax, one pneumomediastinum, and one pneumonia). Tube dislocation was noted in eight patients (11%). Cryobiopsy specimens were significantly larger than forceps biopsy specimens (9.0 mm Conclusions: Thin bronchoscopic cryobiopsy using a nasobronchial tube in consciously sedated patients is safe and effective. Trial registration Date of registration: 24/06/2019. UMIN-Clinical Trials Registry; Identifier: UMIN000037156 https://www.umin.ac.jp/ctr/index.htm.
MeSH term(s)	Biopsy/adverse effects ; Biopsy/methods ; Bronchoscopes ; Bronchoscopy/adverse effects ; Bronchoscopy/methods ; Humans ; Lung/pathology ; Silicones
Chemical Substances	Silicones
Language	English
Publishing date	2022-09-24
Publishing country	England
Document type	Clinical Trial ; Journal Article
ZDB-ID	2059871-3
ISSN	1471-2466 ; 1471-2466
ISSN (online)	1471-2466
ISSN	1471-2466
DOI	10.1186/s12890-022-02166-w
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