Differences in the function and secretion of congenital aberrant fibrinogenemia between heterozygous gamma D320G (Okayama II) and gamma Delta N319-Delta D320 (Otsu I)

Background: We encountered two patients with hypodysfibrinogenemia and designated them as Okayama II and Otsu I. Although the affected residue(s) in Okayama II and Otsu I overlapped, functionally determined fibrinogen levels and the ratio of functionally to immunologically determined plasma fibrinogen levels were markedly different.Methods: DNA sequence and functional analyses were performed for purified plasma fibrinogen. A recombinant protein was synthesized in Chinese hamster ovary (CHO) cells to determine the secretion of variant fibrinogens.Results: A heterozygous A>G in FGG, resulting in gamma 320Asp>Gly for Okayama II, and a heterozygous deletion of AATGAT in FGG, resulting in the deletion of gamma Asn319 and gamma Asp320 (gamma Delta N319-Delta D320) for Otsu I, were obtained. SDS-PAGE and Coomassie staining revealed that the variant gamma-chain was not clear in Okayama II, but was clearly present in Otsu I. The lag period for the fibrin polymerization of Okayama II was slightly slower than that of the normal control, whereas Otsu I fibrinogen indicated no polymerization within 30 min. Both variant gamma-chains were synthesized in CHO cells and assembled into fibrinogen; however, the fibrinogen concentration ratio of the medium/cell lysate of gamma 320Gly was six-fold lower than that of gamma Delta N319-Delta D320.Conclusions: We concluded that the plasma fibrinogen of Okayama II, constituted by a lower ratio of the variant gamma-chain, led to the almost normal functioning of fibrin polymerization. However, the plasma fibrinogen of Otsu I, with a higher ratio of the variant gamma-chain, led to marked reductions in fibrin polymerization. (C) 2015 Elsevier Ltd. All rights reserved.THROMBOSIS RESEARCH. 136(6):1318-1324 (2015)journal articl

Novel heterozygous dysfibrinogenemia, Sumida (A alpha C472S), showed markedly impaired lateral aggregation of protofibrils and mildly lower functional fibrinogen levels

Introduction: We encountered a 6-year-old girl with systemic lupus erythematosus. Although no bleeding or thrombotic tendency was detected, routine coagulation screening tests revealed slightly lower plasma fibrinogen levels, as determined by functional and antigenic measurements (functional/antigenic ratio=0.857), suggesting hypodysfibrinogenemia. Materials and methods: DNA sequence and functional analyses were performed on purified plasma fibrinogen, and recombinant variant fibrinogen was synthesized in Chinese hamster ovary cells based on the results obtained. Results: DNA sequencing revealed a heterozygous A alpha C472S substitution (mature protein residue number) in the alpha C-domain. A alpha C472S fibrinogen indicated the presence of additional disulfide-bonded molecules, and markedly impaired lateral aggregation of protofibrils in spite of slightly lower functional plasma fibrinogen levels. Scanning electron microscopic observations showed a thin fiber fibrin clot, and t-PA and plasminogen-mediated clot lysis was similar to that of a normal control. Recombinant variant fibrinogen-producing cells demonstrated that destruction of the A alpha 442C-472C disulfide bond did not prevent the synthesis or secretion of fibrinogen, whereas the variant A alpha chain of the secreted protein was degraded faster than that of the normal control. Conclusion: Our results suggest that A alpha C472S fibrinogen may cause dysfibrinogenemia, but not hypofibrinogenemia. The destruction and steric hindrance of the alpha C-domain of variant fibrinogen led to the impaired lateral aggregation of protofibrils and t-PA and plasminogen-mediated fibrinolysis, as well as several previously reported variants located in the alpha C-domain, and demonstrated the presence of disulfide-bonded molecules.ArticleTHROMBOSIS RESEARCH. 135(4):710-717 (2015)journal articl

Recombinant gamma T305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole 'a' and calcium binding sites

Introduction: We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio = 0.295), suggesting hypodysfibrinogenemia. Materials and methods: DNA sequence analysis was performed, and gamma T305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant gamma N308K fibrinogen. Results: DNA sequence analysis revealed a heterozygous gamma T305A substitution (mature protein residue number). The gamma T305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1 mM calcium ion compared with that of gamma N308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob 'A') and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole 'a' and D:D interaction sites were all markedly impaired, whereas gamma N308K was impaired at the latter two sites. Molecular modeling demonstrated that gamma T305 is localized at a shorter distance than gamma N308 from the high affinity calcium binding site and hole 'a'. Conclusion: Our findings suggest that gamma T305 might be important for construction of the overall structure of the. module of fibrinogen. Substitution of gamma T305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant gamma T305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.ArticleTHROMBOSIS RESEARCH. 134(2):518-525 (2014)journal articl

Genetic analyses of novel compound heterozygous hypodysfibrinogenemia, Tsukuba I: FGG c.1129+62_65 del AATA and FGG c.1299+4 del A

Epub 2016 Nov 5Introduction: Wefound a novel hypodysfibrinogenemia designated Tsukuba I caused by compound heterozygous nucleotide deletionswith FGG c. 1129+ 62_ 65 del AATA and FGG c. 1299+ 4 del A on different alleles. The former was deep in intron 8 of FGG (IVS-8 deletion) and the latter in exon 9 of FGG (Ex-9 deletion), which is translated for the gamma'-chain, but not the.A-chain. AWestern blot analysis of plasma fibrinogen from our patient revealed an aberrant gamma-chain that migrated slightly faster than the normal B beta-chain. Materials andmethods: To clarify the complex genetic mechanismunderlying Tsukuba I's hypodysfibrinogenemia induced by nucleotide deletions in two regions, we generated two minigenes incorporating each deletion region, transfected them into Chinese Hamster Ovary (CHO) cells, and analyzed RT-PCR products. We also established CHO cells producing the recombinant variant fibrinogen,gamma' 409.A (Ex-9 deletion). Results and conclusions: Minigene I incorporating the IVS-8 deletion showed two products: a normal splicing product and the unspliced product. Minigene II incorporating the Ex-9 deletion only produced the unspliced product. The established gamma' 409.A-CHOcells secreted variant fibrinogenmore effectively than normal fibrinogen. Therefore, the aberrant splicing products derived from the IVS-8 deletion cause hypofibrinogenemia most likely due to nonsense-mediated mRNA decay and the partial production of normal.A-and gamma'-chains; moreover, the Ex-9 deletion causes hypodysfibrinogenemia due to the absence of normal.A-and gamma'-chain production (hypofibrinogenemia) and augmented aberrant.'-chain production (dysfibrinogenemia). (C) 2016 Elsevier Ltd. All rights reserved.ArticleTHROMBOSIS RESEARCH. 148:111-117 (2016)journal articl

An Analysis of Court Cases Relating to Special Education in Japan: Perspective towards Inclusive Education

本稿では，わが国における特別支援教育システムの構築に示唆的な近時の裁判例を要約し，新たな動向を把握することを試みた。具体的には，特別支援教育をめぐる裁判のなかでも，①通常の学校における特別支援教育の問題を争点に含むもの，②過去10年間（2005年以降）に判決が下されたもの，を条件として5つの裁判例を抽出した。それらの裁判における主な経緯や争点の評釈から，通常の学校における特別支援教育が推進されるとともに，多様な教育課題が誘発されており，新たな争点も生じていることが明らかとなった。すなわち，情報開示の範囲，就学先の決定，教員の専門性，学校の情報共有体制といった実践的課題が提起されており，障害のある児童生徒および保護者の権利を重視する判断が示されてきた。「障害者の権利に関する条約」の批准をふまえ，インクルーシブ教育の実現に向けた特別支援教育システムのいっそうの発展が望まれる

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Structure-activity relationship of imidazothiadiazole analogs for the binding to the ecdysone receptor of insect cells.

Diacylhydrazines are the first non-steroidal ecdysone agonists, and five compounds are used as insecticides in agriculture. After the discovery of diacylhydrazine-type compounds, numerous non-steroidal structures were reported as ecdysone agonists. Among various ecdysone agonists, imidazothiadiazoles are reported to be very potent in vitro; however, the experimental detail for the structure identification and bioassays are not stated in the paper (Holmwood and Schindler, Bioorg. Med. Chem. 17, 4064-4070, 2009). In our present study, we synthesized 18 imidazothiadiazole-type compounds and confirmed the chemical structures by spectrometric analyses. The binding activity of the synthesized compounds to the ecdysone receptor was evaluated in terms of the concentration required for 50% inhibition of [(3)H]ponasterone A incorporation [IC50 (M)] into lepidopteran (Sf-9), coleopteran (BCRL-Lepd-SL1), and dipteran (NIAS-AeAl2) cells. 6-(2-Chlorophenyl)-2-(trifluoromethyl)imidazo[2, 1-b] [1, 3, 4]-thiadiazol-5-yl)acrylamide analogs with CONHR (secondary amide) were very potent against Sf-9 cells, but further alkylation (tertiary amide: CONR2) decreased the activity dramatically. Additionally, a primary amide analog (CONH2) was inactive. The activity also decreased 150-fold by the saturation of olefin region of the acrylamide moiety. In addition, various substituents were introduced at the 2-position of the imidazothiadiazole ring to disclose the physicochemical properties of the substituents which are important for receptor binding. The activity increased by 7500-fold with the introduction of the CF2CF2CF3 group compared to the unsubstituted compound against Sf-9 cells. Quantitative structure-activity relationship analysis for these substituents indicated that hydrophobic and electron-withdrawing groups were favorable for binding. Some of the compounds with strong receptor binding activity showed good larvicidal activity against Spodoptera litura. In contrast, the binding affinity of imidazothiadiazole analogs was low or not observed against dipteran and coleopteran cells

Examination of the Impact of Strength and Velocity of the Knee and Ankle on Gait Speed in Community-Dwelling Older Adults

The muscle strength of the knee extension and plantarflexion plays a crucial role in determining gait speed. Recent studies have shown that no-load angular velocity of the lower limb joints is essential for determining gait speed. However, no reports have compared the extent to which lower limb functions, such as knee extension strength, knee extension velocity, plantarflexion strength, and plantarflexion velocity, impact gait speed in a single study. Therefore, this study aimed to examine the relative importance of maximum strength and no-load angular velocity on gait speed. Overall, 164 community-dwelling older adults (72.9 ± 5.0 years) participated in this study. We measured the gait speed and lower limb function (the strength and velocity of knee extension and plantarflexion). Strength was measured with a hand-held dynamometer, and velocity with a gyroscope. A multiple regression analysis was performed with gait speed as the dependent variable and age, sex, and lower-limb function as independent variables. Plantarflexion velocity (β = 0.25) and plantarflexion strength (β = 0.21) were noted to be significant predictors of gait speed. These findings indicate that no-load plantarflexion velocity is more important than the strength of plantarflexion and knee extensions as a determinant of gait speed, suggesting that improvement in plantarflexion velocity may increase gait speed