Identification and Modulation of the Key Amino Acid Residue Responsible for the pH Sensitivity of Neoculin, a Taste-Modifying Protein

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor–taste substance in particular

Comparing α-carbanion-stabilizing ability of substituents using the Brook rearrangement

The α-carbanion-stabilizing ability of the phenylthio and trimethylsilyl groups was compared based on the relative rate of the base-catalyzed Brook rearrangement of the β-substituted α-silylallylalcohol

Characterization of the Modes of Binding between Human Sweet Taste Receptor and Low-Molecular-Weight Sweet Compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2–hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein–coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling–based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands

コウトウ キョウイク カイカク ノ ソウゴ コウイ ブンセキ : ビデオ エスノグラフィー ケンキュウ ノ ネライ ト コウガクブ トシ コウガク エンシュウ ノ ジッサイ

ビデオエスノグラフィーは、当事者的知識を十分に摂取しながら行うビデオ分析であり、我々は この方法で、高等教育改革の現場を研究した。生涯学習社会の到来を受けて、日本の高等教育は現在第 2 次世界大戦直後以来の改革期にある。すなわち、「知識」より「生涯学習能力」の獲得を志向した、 自発性を尊重するような様々な取り組みがなされ始めている。この高等教育の現場に対し、ワークプレ ース研究を行った。Ｂ大学工学部都市工学演習α班を分析対象とした調査の結果、①演習の課題解釈に は「従来の指標の相対化の要求の程度」を巡って２つの解釈があり得たこと、②班内にはその２種類の 解釈に対応した葛藤・対立的相互行為が存在したこと、③にもかかわらず、班内葛藤を生きる当事者が ともに専門性（「都市工学」）を志向していたこと、④したがって、課題理解のいかんにかかわらず、 班活動の全体が「都市工学演習」と呼び得るものになっていたこと、⑤その一方で、最終審査会場（ジ ュリー）ではこの２重性が十分レリバントなものとして浮かび上がって来ていなかったこと、これらの ことがわかった。諸結果を総合すると、学生の自主的活動を尊重するタイプの、新しい学習方法の吟味・ 評価のためには、学生によるその方法の実践状況の分析が有意義であるだろうこと、また、それは、場 合によっては教員の評価のパラダイムを変える力を持つだろうことなどが予測された。なお、本報告は、 文科省科学研究費補助金「高等教育改革のコミュニケーション分析－現場における文化変容の質的検討 －」（基盤研究（Ｂ）、 課題番号 １８３３０１０５、研究代表者：樫田美雄）ほかによる研究成果 の一部である

Use of the index of pulmonary vascular disease for predicting long-term outcome of pulmonary arterial hypertension associated with congenital heart disease

AimsLimited data exist on risk factors for the long-term outcome of pulmonary arterial hypertension (PAH) associated with congenital heart disease (CHD-PAH). We focused on the index of pulmonary vascular disease (IPVD), an assessment system for pulmonary artery pathology specimens. The IPVD classifies pulmonary vascular lesions into four categories based on severity: (1) no intimal thickening, (2) cellular thickening of the intima, (3) fibrous thickening of the intima, and (4) destruction of the tunica media, with the overall grade expressed as an additive mean of these scores. This study aimed to investigate the relationship between IPVD and the long-term outcome of CHD-PAH.MethodsThis retrospective study examined lung pathology images of 764 patients with CHD-PAH aged <20 years whose lung specimens were submitted to the Japanese Research Institute of Pulmonary Vasculature for pulmonary pathological review between 2001 and 2020. Clinical information was collected retrospectively by each attending physician. The primary endpoint was cardiovascular death.ResultsThe 5-year, 10-year, 15-year, and 20-year cardiovascular death-free survival rates for all patients were 92.0%, 90.4%, 87.3%, and 86.1%, respectively. The group with an IPVD of ≥2.0 had significantly poorer survival than the group with an IPVD <2.0 (P = .037). The Cox proportional hazards model adjusted for the presence of congenital anomaly syndromes associated with pulmonary hypertension, and age at lung biopsy showed similar results (hazard ratio 4.46; 95% confidence interval: 1.45–13.73; P = .009).ConclusionsThe IPVD scoring system is useful for predicting the long-term outcome of CHD-PAH. For patients with an IPVD of ≥2.0, treatment strategies, including choosing palliative procedures such as pulmonary artery banding to restrict pulmonary blood flow and postponement of intracardiac repair, should be more carefully considered

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives

Some D-Amino acids such as D-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor

Effect of Dietary Carotenoid on Egg Yolk Color and Singlet Oxygen Quenching Activity of Laying Hens

The effects of dietary carotenoids on egg yolk were investigated in this study. Forty Rhode Island Red (RR) and 40 Silky Fowl (SF) hens that were 60 weeks old were used. Hens of each breed were randomly divided into four dietary groups. One group was fed a basal diet (crude protein 17%, metabolizable energy 2800 kcal/kg) only, whereas the other groups received a specific additive, namely, paprika extract, marigold petal extract, or Paracoccus cell powder, in addition to the same basal diet. The color and carotenoid content of egg yolk and singlet oxygen quenching activity were measured after 4 weeks. The total carotenoid content, zeaxanthin content, and singlet oxygen quenching activity in the yolk differed significantly between breeds and between diets (two-way ANOVA). The lutein content in egg yolk was affected by breed and diet, as well as by the interaction between these two factors. Regarding the Roche Yolk Color Fan values, only the effect of diet was significant. In terms of objective egg yolk color, there was a significant difference in lightness and yellowness between breeds. The total carotenoid content was higher in SF than in RR in all the groups. Likewise, the levels of zeaxanthin and lutein in the yolk were higher in SF than in RR (P<0.05). The results of the present study suggest that dietary carotenoids are effective feed additives for laying hens, especially SF, to improve the color and singlet oxygen quenching activity of egg yolk

An asexual flower of Silene latifolia and Microbotryum lychnidis-dioicae promotes sex-organ development.

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbotryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant without a gynoecium or stamen was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of flower bud development as well as in the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. M. lychnidis-dioicae would have a function similar to stamen-promoting factor (SPF), since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. An infection experiment also revealed that a deletion on the Y chromosome of the asexual mutant eliminated genes for maturation of tapetal cells because the tapetal cells did not mature in the asexual mutant infected with M. lychnidis-dioicae

A bienzyme electrochemical biosensor for the detection of collagen l-hydroxyproline

l-hydroxyproline (l-Hyp: trans-4-hydroxy-l-proline) is commonly found at high concentrations in connective tissue proteins such as collagen. It is a remarkably useful molecular marker because variation in the level of l-Hyp is associated with various diseases. Recently, the novel enzymes l-hydroxyproline epimerase and d-hydroxyproline dehydrogenase were isolated from bacteria. In this study, a novel electrochemical biosensor for l-Hyp was constructed using these two enzymes. l-hydroxyproline epimerase epimerized l-Hyp to d-hydroxyproline (d-Hyp: cis-4-hydroxy-d-proline), and d-Hyp was oxidized with the reaction catalyzed by d-hydroxyproline dehydrogenase and mediated by ferrocene. We found that the sensor could determine l-Hyp concentrations of 10–100 μM with high-selectivity