Publications by authors named "Akira Takagi"

117 Publications

Investigation of the hearing levels of siblings affected by a single GJB2 variant: Possibility of genetic modifiers.

Int J Pediatr Otorhinolaryngol 2021 Oct 12;149:110840. Epub 2021 Jul 12.

Division of Hearing and Balance Research, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo, 152-8902, Japan; Medical Genetics Center, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo, 152-8902, Japan. Electronic address:

Objective: Variants in GJB2 can cause autosomal recessive deafness (DFNB1). There is evidence for genotype-phenotype correlations of GJB2 variants; however, several genotypes can cause varying levels of hearing loss likely attributable to differences in genetic or environmental background. As siblings share approximately 50% of their genetic background and usually have a common environmental background, analysis of phenotypes of siblings with a specific GJB2 variant may reveal factors relevant to phenotypic variation. There have been no previous analyses of differences in hearing among siblings carrying a single GJB2 genotype. Here, we investigated hearing differences between siblings with a single GJB2 variant, which can cause various levels of hearing loss.

Methods: We examined hearing levels in 16 pairs of siblings homozygous for the c.235delC variant of GJB2. Differences in hearing acuity between sibling pairs were detected by auditory evaluation.

Results: Average differences in acoustic threshold >30 dB were observed between five pairs of siblings, whereas the remaining 11 pairs had average threshold values within approximately 10 dB of one another. Hearing loss varied from moderate to profound.

Conclusion: Our results indicate that auditory acuity associated with homozygosity for GJB2 c.235delC can vary in degree; however, in approximately 70% of younger siblings, it was approximately the same as that in the first child, despite a diverse spectrum of hearing loss among different families. These results suggest that differences in genetic background may modify the phenotype associated with homozygous GJB2 c.235delC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijporl.2021.110840DOI Listing
October 2021

Could London Dispersion Force Control Regioselective (2 + 2) Cyclodimerizations of Benzynes? YES: Application to the Synthesis of Helical Biphenylenes.

J Am Chem Soc 2021 Jul 1;143(29):10853-10859. Epub 2021 Jul 1.

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

In recent years, London dispersion interactions, which are the attractive component of the van der Waals potential, have been found to play an important role in controlling the regio- and/or stereoselectivity of various reactions. Particularly, the dispersion interactions between substrates and catalysts (or ligands) are dominant in various selective catalyzes. In contrast, repulsive steric interactions, rather than the attractive dispersion interactions, between bulky substituents are predominant in most of the noncatalytic reactions. Herein, we demonstrate the first example of London dispersion-controlled noncatalytic (2 + 2) cyclodimerization of substituted benzynes to selectively afford proximal biphenylenes in high yields and regioselectivities, depending on the extent of dispersion interactions in the substituents. This method can be applied for the synthesis of novel helical biphenylenes, which would be fascinating for chemists as these compounds are potential skeletons for ligands, catalysts, and medicines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.1c05434DOI Listing
July 2021

Esophageal inflammatory pseudotumor with low-dose corticosteroid therapy after surgery.

Clin J Gastroenterol 2021 Apr 7;14(2):427-433. Epub 2021 Mar 7.

Department of Gastroenterological Surgery, Shizuoka General Hospital, 4-27-1 Kita Ando Aoi-ku, Shizuoka City, 420-8527, Japan.

Inflammatory pseudotumors of the esophagus are extremely rare, and the treatment has been controversial. Herein, we report a case of esophageal inflammatory pseudotumor with low-dose corticosteroid treatment following surgery. A 50-year-old woman with a 3-month history of progressive dysphagia and weight loss, was admitted to our hospital for examination and treatment. Esophagography and endoscopic examination revealed a mass present from the cervical esophagus to the upper thoracic esophagus with severe esophageal stricture. Ultrasound-guided fine needle aspiration cytology, boring biopsy, and mucosal incision-assisted biopsy reveal chronic inflammation, but histological diagnosis was not proven. Surgery was performed to confirm diagnosis and to relieve esophageal stricture. However, because of dense adhesions around the tumor, complete tumor resection was not achieved. Histopathological examination showed an inflammatory infiltrate with plasma cells, eosinophils, neutrophils, and lymphocytes, suggesting an inflammatory pseudotumor. After surgical resection, the esophageal stricture remained, possibly due to the residual tumor. We used a postoperative low-dose steroid treatment that resulted in complete resolution. There has not been any evident sign of recurrence for more than 2 years.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12328-021-01377-8DOI Listing
April 2021

Total Synthesis of Antiausterity Agent (±)-Uvaridacol L by Regioselective Axial Diacylation of a -Inositol Orthoester.

Org Lett 2021 Jun 17;23(11):4083-4087. Epub 2021 Feb 17.

Laboratory of Bioorganic & Natural Products Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada, Kobe, Hyogo 658-8558, Japan.

The antiausterity natural product (±)-uvaridacol L was synthesized for the first time in seven steps from -inositol. The key reaction of this synthesis, axial selective dibenzoylation of -inositol orthoformate, was achieved using a catalytic amount of tetrabutylammonium fluoride (TBAF). The preferential cytotoxicity of racemic uvaridacol L against cancer cell lines able to adapt to nutrient deprivation was also evaluated under nutrient deprived conditions. Morphological evaluation was also carried out.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.orglett.1c00079DOI Listing
June 2021

Essential role of a carboxyl-terminal α-helix motif in the secretion of coagulation factor XI.

J Thromb Haemost 2021 04 10;19(4):920-930. Epub 2021 Feb 10.

Division of Cellular and Genetic Sciences, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Background: Coagulation factor XI (FXI) is a plasma serine protease zymogen that contributes to hemostasis. However, the mechanism of its secretion remains unclear.

Objective: To determine the molecular mechanism of FXI secretion by characterizing a novel FXI mutant identified in a FXI-deficient Japanese patient.

Patient/methods: The FXI gene (F11) was analyzed by direct sequencing. Mutant recombinant FXI (rFXI) was overexpressed in HEK293 or COS-7 cells. Western blotting and enzyme-linked immunosorbent assay were performed to examine the FXI extracellular secretion profile. Immunofluorescence microscopy was used to investigate the subcellular localization of the rFXI mutant.

Results: We identified a novel homozygous frameshift mutation in F11 [c.1788dupC (p.E597Rfs*65)], resulting in a unique and extended carboxyl-terminal (C-terminal) structure in FXI. Although rFXI-E597Rfs*65 was intracellularly synthesized, its extracellular secretion was markedly reduced. Subcellular localization analysis revealed that rFXI-E597Rfs*65 was abnormally retained in the endoplasmic reticulum (ER). We generated a series of C-terminal-truncated rFXI mutants to further investigate the role of the C-terminal region in FXI secretion. Serial rFXI experiments revealed that a threonine at position 622, the fourth residue from the C-terminus, was essential for secretion. Notably, Thr622 engages in the formation of an α-helix motif, indicating the importance of the C-terminal α-helix in FXI intracellular behavior and secretion.

Conclusion: FXI E597Rfs*65 results in the pathogenesis of a severe secretory defect resulting from aberrant ER-to-Golgi trafficking caused by the lack of a C-terminal α-helix motif. This study demonstrates the impact of the C-terminal structure, especially the α-helix motif, on FXI secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15242DOI Listing
April 2021

Identification of HLA-A*02:01-restricted candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 that may be natural targets of CD8 T cell recognition .

J Virol 2020 Dec 2. Epub 2020 Dec 2.

Department of Microbiology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-γ-producing CD8 T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three response patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. For the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three reaction patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01837-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092843PMC
December 2020

Incidence of Mumps Deafness in Japan, 2005-2017: Analysis of Japanese Insurance Claims Database.

J Epidemiol 2020 Oct 24. Epub 2020 Oct 24.

Department of Pediatrics, Shizuoka Kosei Hospital.

Background: Mumps deafness causes serious problems, and incidence data are needed to identify its disease burden. However, such data are limited, and the reported incidence is highly variable. Nationwide studies in Japan with a large age range are lacking.

Methods: This was a retrospective observational investigation of the 2005-2017 mumps burden using employment-based health insurance claims data. Data were analyzed for 5,190,326 people aged 0-64 years to estimate the incidence of mumps deafness.

Results: Of 68,112 patients with mumps (36,423 males; 31,689 females), 102 (48 males; 54 females) developed mumps deafness-an incidence of 15.0 per 10,000 patients (1 in 668 patients). Fifty-four (52.9%) patients had mumps deafness in childhood (0-15 years), and 48 (47.1%) had mumps deafness in adolescence and adulthood (16-64 years); most cases occurred in childhood, the peak period for mumps onset. The incidence of mumps deafness per 10,000 patients was 73.6 in adolescence and adulthood, 8.4 times higher than the incidence of 8.8 in childhood (P < 0.001). In childhood, the incidence of mumps deafness was 7.2 times higher among 6-15-year-olds (13.8 [95% CI, 10.2-18.2]) than among 0-5-year-olds (1.9 [95% CI, 0.6-4.5]), and this difference was statistically significant (P < 0.001). No sex difference was observed.

Conclusions: The incidence of mumps deafness per 10,000 patients aged 0-64 years was 15.0 (1 in 668 patients). A secondary risk of deafness following mumps virus infection was identified not only for children, but also for adolescents and adults.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2188/jea.JE20200233DOI Listing
October 2020

A fluorogenic probe using a catalytic reaction for the detection of trace intracellular zinc.

Chem Commun (Camb) 2020 Nov 16;56(87):13327-13330. Epub 2020 Oct 16.

Laboratory of Bioorganic & Natural Products Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama-kita, Higashinada, Kobe 658-8558, Japan.

Labile zinc plays various roles in cells at low concentrations which most fluorescent probes are not able to detect. Here we report a cephem-based probe which coordinates to zinc and zinc-bound water cleaves the scaffold and releases the fluorophore. In addition, the zinc is recycled and reacts with multiple probes, amplifying the signal. This signal amplification system is useful for the detection of intracellular zinc at low concentrations and has potential for further development of probes with a similar molecular design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cc05315eDOI Listing
November 2020

Mumps-Related Disease Burden in Japan: Analysis of JMDC Health Insurance Reimbursement Data for 2005-2017.

J Epidemiol 2021 Aug 13;31(8):464-470. Epub 2021 Jan 13.

Department of Pediatrics, Shizuoka Kosei Hospital.

Background: Mumps vaccination coverage is low in Japan, partly because of its voluntary nature. Although pediatric cases of mumps virus infection are captured by the National Epidemiological Surveillance of Infectious Diseases program under the Infectious Disease Law, there are currently no data regarding the occurrence of mumps and its complications in adults.

Methods: We investigated the annual incidence rates of mumps and its complications based on health insurance reimbursement data for 5,209,660 individuals aged 0-64 years for 2005-2017, obtained from JMDC Inc., to estimate the mumps-related disease burden during this period.

Results: There were three mumps outbreaks (2006, 2010, and 2016) during 2005-2017. The annual incidence of mumps was highest in individuals aged 0-5 years (808-3,792 per 100,000 persons), followed by those aged 6-15 years (658-2,141 per 100,000 persons). The incidence of mumps was higher in females than in males (male/female ratio, 0.90). Among mumps-related complications, the overall incidence (per 1,000 mumps cases) was highest for orchitis (6.6), followed by meningitis (5.8), deafness (1.3), pancreatitis (0.5), and encephalitis (0.3). No cases of oophoritis were noted. The overall incidence of mumps-related complications was 2.5 times higher in males than in females.

Conclusions: This study revealed the disease burden due to mumps and its complications in Japan during 2005-2017. These data suggest the need for mumps-prevention measures in adolescents and adults, as well as in children.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2188/jea.JE20200048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8275443PMC
August 2021

Aberrant X chromosomal rearrangement through multi-step template switching during sister chromatid formation in a patient with severe hemophilia A.

Mol Genet Genomic Med 2020 09 5;8(9):e1390. Epub 2020 Jul 5.

Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Background: Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by pathogenic variants of the coagulation factor VIII gene (F8). Half of the patients with severe HA have a recurrent inversion in the X chromosome, that is, F8 intron 22 or intron 1 inversion. Here, we characterized an abnormal F8 due to atypical complex X chromosome rearrangements in a Japanese patient with severe HA.

Methods: Recurrent F8 inversions were tested with inverse shifting-PCR. The genomic structure was investigated using PCR-based direct sequencing or quantitative PCR.

Results: The proband's X chromosome had a 119.5 kb insertion, a reverse duplex of an extragenic sequence on the F8 telomere region into the F8 intron 1 with two breakpoints. The telomeric breakpoint was a joining from the F8 intron 1 to the inverted FUNDC2 via a two-base microhomology, and the centromeric breakpoint was a recombination between F8 intron 1 homologous sequences. The rearrangement mechanism was suggested as a multi-step rearrangement with template switching such as fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR) and/or homologous sequence-associated recombination during a sister chromatid formation.

Conclusion: We identified the aberrant X chromosome with a split F8 due to a multi-step rearrangement in a patient with severe HA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mgg3.1390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507428PMC
September 2020

Major and trace elements in the surface water of Tonle Sap Lake, Mekong River, and other tributary rivers in Cambodia.

Environ Monit Assess 2020 Jun 29;192(7):467. Epub 2020 Jun 29.

Fisheries administration of Ministry of Agriculture Forestry and Fisheries, 186, Norodom Blvd, Sangkat, Tonle Bassac, Khan Chamcarmon, Phnom Penh, P.O box 582, Cambodia.

To evaluate the seasonal water circulation of Tonle Sap Lake and its tributary rivers in Cambodia, the spatial distribution patterns of major and trace elements in surface water were investigated. Based on the similarity of the dissolved elemental concentrations, the water samples were mainly divided into the three groups: samples with relatively high percentages of Ca, Mo, and Sb (Subcluster B1); samples with high Si, Al, and Fe (B2); and samples with high Na, K, and Mg (B3). During the rainy season, the elemental composition of lake water (B1) appeared to be greatly influenced by the intrusion of water from the Mekong River (B1) through the Tonle Sap River (B1). During the dry season, the type of lake water shifted to B3, suggesting that the lake water stored during the rainy season was replaced by inflow from other tributaries and groundwater in its vicinity. Thus, the seasonal changes in the elemental composition of the lake water were largely controlled by surface water and groundwater circulation. The dissolved As concentration was higher in the lake water and during the dry season than that in the river water and during the rainy season, indicating the discharge of As from the lake's bottom sediment during the dry season. Although the redox cycling of Fe and Mn appeared to be less important due to the shallow water depth in the lake, there are potential risks of As poisoning induced by the formation of an anoxic water mass and increment in the concentration of phosphorus if eutrophication continues to progress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10661-020-08292-4DOI Listing
June 2020

One-Pot Generation of Functionalized Benzynes from Readily Available 2-Hydroxyphenylboronic Acids.

J Org Chem 2020 03 17;85(5):3383-3392. Epub 2020 Feb 17.

Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita, Osaka 565-0871, Japan.

We developed a one-pot method for the generation of benzynes from a range of readily available 2-hydroxyphenylboronic acids. This method features the in situ activation of both boronic acid and hydroxyl groups of the substrate to enhance benzyne generation at 60 °C. Such mild conditions facilitate the generation of functionalized benzynes that immediately react with diverse arynophiles to produce multisubstituted fused benzenes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.joc.9b03169DOI Listing
March 2020

Correlation of indoleamine-2,3-dioxigenase 1 inhibitory activity of 4,6-disubstituted indazole derivatives and their heme binding affinity.

Bioorg Med Chem Lett 2019 10 7;29(19):126607. Epub 2019 Aug 7.

Laboratory of Synthetic Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita, Osaka 565-0871, Japan. Electronic address:

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that acts on the first and rate-limiting step of the tryptophan/kynurenine pathway. Since the pathway is one of the means of cancer immune evasion, IDO1 inhibitors have drawn interest as potential therapeutics for cancers. We found a 4,6-disubstituted indazole 1 as a hit compound that showed both IDO1 inhibitory activity and binding affinity for IDO1 heme. Structural modification of 1 yielded compound 6, whose relatively large substituent at the 4-position and proper size substituent at the 6-position were found to be important for the enhancement of IDO1 inhibitory activity and heme affinity. A series of compounds synthesized in this work were evaluated by in silico docking simulations and by in vitro experiments using a C129Y mutant of the pocket-A of IDO1. Our results revealed that proper substituents at the 6- and 4-positions of the compounds interact with pockets A and B, respectively, and that, in particular, a good fit in pocket-A is important for the compounds' biological activities. Absorption spectral analysis of these compounds showed that they strongly bound to the ferrous heme rather than its ferric heme. Furthermore, we observed that the heme affinities of these compounds strongly correlate with their IDO1 inhibitory activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2019.08.011DOI Listing
October 2019

Apparent synonymous mutation F9 c.87A>G causes secretion failure by in-frame mutation with aberrant splicing.

Thromb Res 2019 Jul 1;179:95-103. Epub 2019 May 1.

Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address:

Introduction: Hemophilia B is an X-linked recessive bleeding disorder caused by coagulation factor IX (FIX) gene (F9) mutations. Several F9 synonymous mutations have been known to cause hemophilia B; however, the deleterious mechanisms underlying the development of hemophilia B have not been completely understood. To elucidate the molecular pathogenesis causing hemophilia B, we investigated the synonymous F9 mutation: c.87A>G, p.(Thr29=).

Materials And Methods: The influence of F9 c.87A>G on mRNA splicing was analyzed by exon-trap assay and in silico prediction. We prepared FIX expression vectors using mutant F9 cDNA and transfected HepG2 cells to investigate intracellular transport and extracellular secretion of FIX. Intracellular kinetics of the expressed FIX was examined by treatment with the proteasome inhibitor MG132.

Results: Exon-trap analysis revealed that F9 c.87A>G resulted in almost (99.1%) aberrant splicing (r.83_88del). In silico analysis predicted that F9 c.87A>G influenced the splicing pattern by generating an available aberrant 5' splice site. The aberrant F9 mRNA (r.83_88del) was translated to a mutant FIX p.Cys28_Val30delinsPhe with an in-frame mutation at the signal peptide cleavage site. Simultaneously, a small amount (0.9%) of mutant F9 r.87A>G translating into WT FIX p.Thr29 = was also observed. The mutant FIX was abnormally retained in the endoplasmic reticulum (ER) and was not extracellularly secreted. It appeared to be intracellularly degraded via proteasome-dependent degradation machinery.

Conclusion: F9 c.87A>G was found to cause abnormal mRNA splicing, r.83_88del, and produce the mutant FIX p.Cys28_Val30delinsPhe. The mutant FIX is an abnormal protein with extracellular secretory defects and is intracellularly eliminated via proteasome-dependent ER-associated degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2019.04.022DOI Listing
July 2019

Molecular basis of SERPINC1 mutations in Japanese patients with antithrombin deficiency.

Thromb Res 2019 Jun 11;178:159-170. Epub 2019 Apr 11.

Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address:

Background: Congenital antithrombin (AT) deficiency, which arises from various SERPINC1 defects, is an autosomal-dominant thrombophilic disorder associated with a high risk of recurrent venous thromboembolism.

Patients/methods: We investigated SERPINC1 defects in Japanese patients with congenital AT deficiency who developed venous thromboembolism or had a family history of deep vein thrombosis. We analyzed the full DNA sequences of SERPINC1 exons and exon-intron junctions by PCR-mediated direct sequencing. If no mutation was found, multiplex ligation-dependent probe amplification (MLPA) was conducted for the relative quantification of the copy number of all exons in SERPINC1. If splice-site mutations were detected, mRNA splicing abnormalities were further investigated using an in vitro cell-based exontrap assay.

Results: We identified 19 different SERPINC1 abnormalities, including 8 novel mutations, in 21 Japanese patients with AT deficiency. These abnormalities were distributed as follows: 9 missense mutations (42.9%), 3 nonsense mutations (14.3%), 1 splice-site mutation (4.8%), 2 small insertions (9.5%), 2 deletion mutations (9.5%) and 4 large deletions (19.0%). Cases with large deletions of SERPINC1 included Alu-mediated gene rearrangements and non-Alu-mediated complex gene rearrangements; the latter could conceivably be explained using the fork stalling and template switching (FoSTeS) model.

Conclusions: We identified a variety of SERPINC1 defects in Japanese patients with AT deficiency. The SERPINC1 mutations detected in patients with type I AT deficiency included single nucleotide missense or nonsense mutations, small intragenic insertions or deletions, and large genomic structural deletions. Large deletions of SERPINC1 were caused by various recurrent or non-recurrent complex genomic rearrangement mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2019.04.004DOI Listing
June 2019

Familial pulmonary thromboembolism with a prothrombin mutation and antithrombin resistance.

J Cardiol Cases 2018 Jun 27;17(6):197-199. Epub 2018 Mar 27.

Department of Cardiovascular Medicine, The Jikei University School of Medicine, Tokyo, Japan.

We describe the case of a 45-year-old man with congenital thrombophilia induced by antithrombin resistance. He had recurrent venous thrombosis without traditional risk factors or abnormal coagulation function and had a family history of venous thrombosis which included his mother, brother, and nephew. We suspected the association of hereditary antithrombin resistance, which has been reported in some cases of familial venous thromboses due to prothrombin mutations. Although prothrombin abnormality typically shows a bleeding tendency, variations of arginine at position 596 in the gene encoding prothrombin have been reported to conversely cause thrombosis. Therefore, we tested and detected antithrombin resistance in the patient's plasma. We also performed genetic analysis for his second filial generation, and found a missense mutation (c.1787G>A), resulting in a substitution of arginine for glutamine at position 596 (p.Arg596Gln) in the gene encoding prothrombin (called prothrombin Belgrade). The Gln596 substitution caused the susceptibility to thrombosis. This variation is the same as one previously reported in Serbia and India, and it is the third report in Japan. < Venous thrombosis is caused by multiple factors. However, approximately 20% of the patients with venous thrombosis of unknown cause have no risk factors. The present case had no known risk factors, rather he has a family history of thrombosis. Therefore, we suspected an association with inherited thrombophilia. Although several gene mutations have been found in some families with inherited thrombophilia, many mutations remain to be detected.>.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jccase.2018.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149589PMC
June 2018

Stapes surgery preserving the superstructure of stapes (Takagi's stapedotomy) in otosclerosis: A retrospective study of 24 consecutive cases.

Auris Nasus Larynx 2018 Dec;45(6):1178-1182

Department of Otolaryngology, Kurashiki Central Hospital, 1-1-1 Miwa, Kurashiki City, Okayama Prefecture, 710-8602, Japan. Electronic address:

Objective: The aim of this study was to evaluate the hearing outcomes and complications of stapedotomy in which the stapes superstructure was preserved (Takagi's stapedotomy). In this surgical approach, the lenticular process of the incus rather is removed, than the superstructure of the stapes.

Methods: A single-center retrospective observational study was performed. We included all patients having Takagi's stapedotomy for otosclerosis between January 2005 and April 2016. Both primary and revision stapes surgery were included. We evaluated audiometric outcomes and surgical complications.

Results: Twenty-four patients who underwent stapedotomy preserving superstructure were included in this study. The postoperative air-bone gap at 1year postoperatively was ≤10dB in 66.7% of patients and ≤20dB in all cases. In longer follow-up period, elevation of the air-bone gap was not observed over the 5 postoperative years in available cases. The postoperative air-bone gap was ≤10dB in 72.2% at 3years and 81.8% at 5years postoperatively.

Conclusion: Takagi's stapedotomy restore ossicular conduction without the removal of superstructure of stapes. The air-bone gap did not get worse in long-term follow-up, although audiometric results would be unsatisfactory. Further larger studies are needed to evaluate the efficacy and safety of Takagi's stapedotomy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.anl.2018.05.004DOI Listing
December 2018

Vwf K1362A resulted in failure of protein synthesis in mice.

Int J Hematol 2018 Apr 1;107(4):428-435. Epub 2018 Feb 1.

Department of Pathology and Clinical Laboratories, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells (ECs) and has two main roles: to carry and protect coagulation factor VIII (FVIII) from degradation by forming VWF-FVIII complex; and to mediate platelet adhesion and aggregation at sites of vascular injury. Previous research using the HEK293 cell line revealed that the VWF K1362 mutation interacted directly with platelet glycoprotein Ib (GPIb). Vwf K1362A knock-in (KI) mice were therefore generated to verify the in vivo function of residue 1362 in binding to platelet GPIb. The Cre-loxP system was employed to introduce the Vwf K1362A mutation systemically in mice. In blood coagulation analysis, the VWF antigen (VWF:Ag) of Lys1362Ala KI homozygous (homo) mice was below the sensitivity of detection by enzyme-linked immunosorbent assay. FVIII activities (FVIII:C) were 47.9 ± 0.3 and 3.3 ± 0.3% (K1362A heterozygous (hetero) and K1362A KI homo mice, respectively) compared to wild-type mice. Immunohistochemical staining analysis revealed that VWF protein did not exist in ECs of K1362A KI homo mice. These results indicated that VWF protein synthesis of K1362A was impaired after transcription in mice. K1362 seems to represent a very important position not only for VWF function, but also for VWF synthesis in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12185-017-2394-yDOI Listing
April 2018

[Development of Efficient Methods for Benzyne Generation].

Authors:
Akira Takagi

Yakugaku Zasshi 2018 ;138(1):27-35

Graduate School of Pharmaceutical Sciences, Osaka University.

 2-[(Neopentyl glycolato)boryl]phenyl triflates, readily synthesized from 2-iodophenol derivatives via halogen-magnesium exchange or Miyaura borylation, were developed as new benzyne precursors. Benzynes were generated under fluoride-ion-mediated conditions and reacted immediately with various arynophiles. Herein, we describe the generation of benzynes having reactive functional groups, such as methoxycarbonyl, acetyl, bromo, and amino groups, as well as their [4+2], (3+2), and [2+2] cycloaddition reactions which produce corresponding benzo-fused compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1248/yakushi.17-00157DOI Listing
February 2018

In vitro exploration of latent prothrombin mutants conveying antithrombin resistance.

Thromb Res 2017 Nov 20;159:33-38. Epub 2017 Sep 20.

Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address:

Introduction: Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599).

Materials And Methods: We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma.

Results: The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19).

Conclusions: Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2017.09.020DOI Listing
November 2017

Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes.

Biochem Biophys Res Commun 2017 10 14;492(1):27-32. Epub 2017 Aug 14.

Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan. Electronic address:

Several non-radioactive methods have widely been utilized to detect antigen-specific cytotoxic T lymphocyte (CTL) responses instead of the classical Cr-release assay. These methods include intracellular cytokine staining, major histocompatibility complex-class I tetramers, and the CD107a mobilization assay. However, they do not directly measure target-cell death. In contrast, several attempts have been made to develop the flow cytometric CTL (FC-CTL) assay for evaluation of cytotoxicity. However, further improvement is necessary for it to become standardized. Here, we evaluated the characteristics of the FC-CTL assay based on the uptake of propidium iodide (PI) using target cell lines expressing the green fluorescent protein (GFP). The FC-CTL assay was found to be sensitive enough to detect primary CTL responses. The usage of a pre-established GFP-expressing target cell line facilitated the procedure of the assay, and enabled a clear discrimination between target and effector cells. Time-course analyses demonstrated that PI-stained target cells were detected as early as surface CD107a expression after antigenic stimulation. Thus, the PI/GFP-based FC-CTL assay is sufficiently sensitive to practically detect the early stages of target-cell death, and may have a great potential for becoming a standard tool to measure CTL activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2017.08.045DOI Listing
October 2017

Three-Dimensional Printing Model as a Tool to Assist in Surgery for Large Mandibular Tumour: a Case Report.

J Oral Maxillofac Res 2017 Apr-Jun;8(2):e4. Epub 2017 Jun 30.

Department of Dentistry, Oral and Maxillofacial - Plastic and Reconstructive Surgery Faculty of Medicine, Yamagata University, YamagataJapan.

Objectives: Recently, three-dimensional printing models based on preoperative computed tomography and magnetic resonance imaging images have been widely used in medical fields. This study presents an effective use of the three-dimensional printing model in exploring complex spatial relationship between the tumour and surrounding tissue and in simulation surgery based planning of the operative procedure.

Material And Methods: The patient was a 7-year-old boy with ameloblastic fibro-odontoma. Prior to surgery, a hybrid three-dimensional printing model consisting of the jaw bone, the tumour and the inferior alveolar nerve was fabricated. After the simulation surgery based on this model, enucleation of the tumour, leaving tooth 46 intact (Universal Numbering System by ADA) safe, was planned.

Results: Enucleation of the tumour was successfully carried out. One year later, healing was found to be satisfactory both clinically and radiographically.

Conclusions: The study presented an effective application of a novel hybrid three-dimensional printing model composed of hard and soft tissues. Such innovations can bring significant benefits, especially to the field of oncological surgery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5037/jomr.2017.8204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541989PMC
June 2017

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

Thromb Haemost 2016 Nov 8;116(6):1022-1031. Epub 2016 Sep 8.

Tetsuhito Koijima, Nagoya University Graduate School of Medicine, Pathophysiological Laboratory Sciences, 1-1-20 Daiko-Minami Higashi-ku, Nagoya Aichi 461-8673, Japan, Tel.: +81 52 719 3153, Fax: +81 52 719 3153, E-mail:

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1160/TH16-03-0223DOI Listing
November 2016

1,3- and 1,4-Benzdiyne equivalents for regioselective synthesis of polycyclic heterocycles.

Chem Sci 2016 Aug 18;7(8):5206-5211. Epub 2016 Apr 18.

Graduate School of Pharmaceutical Sciences , Osaka University , 1-6 Yamadaoka , Suita , Osaka 565-0871 , Japan . Email: ; Email:

We have devised a novel 1,3-benzdiyne equivalent, capable of quadruple functionalization by sequential benzyne generation and reaction with arynophiles. The key features of this method include the chemoselective generation of two triple bonds in a single benzene ring under fluoride-mediated mild conditions, and the regiocontrol of each benzyne reaction by the substituent next to the triple bond. This method produced various benzo-fused heteroaromatic compounds reactions with arynophiles, such as furans, azides, and diazo compounds. A validation of the method is given in the convergent synthesis of the antipsychotic drug risperidone. A similar strategy has also been applied to a 1,4-benzdiyne equivalent to construct linearly benzo-fused heteroaromatics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c6sc00798hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020534PMC
August 2016

Progestin isoforms provide different levels of protein S expression in HepG2 cells.

Thromb Res 2016 Sep 16;145:40-5. Epub 2016 Jul 16.

Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Japan. Electronic address:

Introduction: Use of combined oral contraceptives (COCs) results in acquired protein S (PS) deficiency, a well-established risk factor for venous thromboembolism (VTE). The risk of VTE due to COCs containing newer-generation progestins is double compared with COCs containing older-generation progestins, although there is little difference in estrogen contents between the generations. In contrast, progestin-only contraceptives do not confer an increased risk of VTE. In this study, we aimed to investigate how different isoforms of progestin in COCs affect the risk of VTE by measuring PS expression.

Materials And Methods: The effect of progestin, levonorgestrel (LNG) or drospirenone (DRSP), on PS mRNA expression in HepG2 cells was measured using reverse transcription-quantitative PCR; PS level was determined using Western blot analysis. PROS1 promoter activity, PS mRNA stability, and de novo synthesis of PS mRNA were examined in HepG2 cells after treatment with progestin.

Results And Conclusions: In the presence of progestins, PS mRNA and protein expressions were significantly upregulated in HepG2 cells due to the augmentation of de novo PS mRNA expression modulated by RNA polymerase II (Pol II), thereby facilitating PS transcription elongation. Moreover, the transcription elongation inhibitor blocked progestin-mediated de novo PS mRNA expression. Conversely, progestin did not affect PROS1 promoter activity and PS mRNA stability. Pol II elongation efficiency in the newer-generation progestin (DRSP) treatment was not as strong compared with older-generation progestin (LNG), suggesting the difference in VTE risk between COC generations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2016.07.007DOI Listing
September 2016

Safety of heat generated by endoscope light sources in simulated transcanal endoscopic ear surgery.

Auris Nasus Larynx 2016 Oct 21;43(5):501-6. Epub 2016 Jan 21.

Department of Otolaryngology, Head and Neck Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata-shi, Yamagata 990-9585, Japan.

Objective: To determine whether heat generated by endoscope light sources during ear surgery is safe.

Methods: Transcanal endoscopic ear surgery (TEES) was simulated using 2.7-mm or 4-mm endoscopes coupled to xenon or LED light sources and a 3D model of human temporal bone. The endoscope tip was fixed at the center of tympanic annulus. Light sources were tested at clinical (30% for xenon and 40% for LED) and 100% settings. Temperatures were measured using thermocouples attached to the endoscope tip and three points within the middle ear cavity: promontory, horizontal portion of the facial nerve and lateral semicircular canal.

Results: Maximum temperatures measured within the middle ear cavity were below 31°C at clinical settings, while the temperatures rose to 44.1°C using a 4-mm endoscope with a xenon light source set at 100%. Temperatures measured at the tip were all safe at clinical settings, but rose dramatically to 110.1°C for the 4-mm endoscope with xenon at 100%.

Conclusion: Endoscopes can be safely used within the middle ear at clinical settings. However, operators should not exceed clinical settings, particularly with 4-mm endoscopes with a xenon light source, to ensure temperatures generated within the middle ear cavity are safe.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.anl.2015.12.014DOI Listing
October 2016
-->