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Background Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous neuromuscular disorder characterized by muscle weakness and caused by mutations in more than 35 different genes. This condition should not be overlooked as a subset of patients with CMS are treatable. However, the diagnosis of CMS is often difficult due to the broad variability in disease severity and course. Case report A five-year-old boy without remarkable family history was born with marked general muscle hypotonia and weakness, respiratory insufficiency, anomalies, and multiple joint contractures. Congenital myopathy was suspected based upon type 1 fiber predominance on muscle biopsy. However, he was diagnosed with CMS at age 4 years when his ptosis and ophthalmoplegia were found to be improved by edrophonium chloride and repetitive nerve stimulation showed attenuation of compound muscle action potentials. An exome sequencing identified a compound heterozygous missense variant of c.737C > T (p.A246V) and a novel intronic insertion c.1166 + 4_1166 + 5insAAGCCCACCAC in RAPSN. RT-PCR analysis which showed the skipping of exon 7 in a skeletal muscle sample confirmed that the intronic insertion was pathogenic. His myasthenic symptoms were remarkably improved by pyridostigmine. Conclusion The patient’s diagnosis of CMS was confirmed by exome sequencing, and RT-PCR revealed that the skipping of exon 7 in RAPSN was caused by a novel intronic insertion. The genetic information uncovered in this case should therefore be added to the collection of tools for diagnosing and treating CMS.

Active collagen oligopeptides (ACOP) are bioactive collagen-derived peptides detected by a recently-established ELISA. To facilitate studies of the function and metabolism of these products, this study aims to determine which of these peptides is recognized by a novel anti-ACOP antibody used in this ELISA. We then investigate the effect of collagen peptide (CP) ingestion and exercise on urinary ACOP concentrations in a cohort of university student athletes using colorimetric, LC–MS/MS, and ELISA. We observed that the antibody showed strong cross-reactivity to Pro-Hyp and Gly-Pro-Hyp and weak cross-reactivity to commercial CP. CP ingestion increased the urinary level of ACOP over time, which correlated highly with urinary levels of peptide forms of Hyp and Pro-Hyp. Physical activity significantly decreased the urinary ACOP level. This study demonstrates changes in urinary ACOP following oral CP intake and physical activity using ELISA with the novel anti-ACOP antibody. Thus, ACOP may be useful as a new biomarker for collagen metabolism., Article number: 13527 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

The inclusion complexations of rhodamine derivatives with native and 2,6-di-O-methylated β-cyclodextrins (β-CD and DM-β-CD) were studied spectrophotometrically. Rhodamine derivatives were shown to form 1:1 inclusion complexes with β-CDs by the continuous variation method. The structures of the inclusion complexes were characterized by 1H-1H rotating frame nuclear Overhauser effect spectroscopy (ROESY) NMR measurements. It was found that native β-CD encapsulates the xanthenyl ring of rhodamines into the cyclodextrin cavity, while DM-β-CD forms two group-in complexes (phenyl-in and xanthenyl-in (bidirectional (bimodal) inclusion complexes)) with rhodamines bearing moderately bulky functional groups. Furthermore, we demonstrated the unique thermodynamics for the group-inclusion complex formation by DM-β-CD. The quantum yields for the inclusion complexes of rhodamines were determined using a quantum measurement apparatus equipped with a half-moon unit. The results indicated that the cyclodextrin inclusion of rhodamines with the bulky amino substituents on the xanthenyl ring largely decreases the quantum yield values. Based on these results, the substituent effects on the fluorescence process for the cyclodextrin inclusion complexes of rhodamines were discussed. This study provides useful insights for the functional group recognition of native and modified β-CDs.

Collagen-derived dipeptide prolyl-hydroxyproline (Pro-Hyp) directly binds to the forkhead box g1 (Foxg1) protein and causes it to undergo structural alteration. Pro-Hyp also promotes the production of a regulator of osteoblast differentiation, Runt-related transcription factor 2 (Runx2), through Foxg1, inducing osteoblast differentiation. In addition, Pro-Hyp disrupts the interaction between Foxg1 and Runx2, and Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp. To elucidate the mechanism of Pro-Hyp that promotes osteoblast differentiation, we investigated whether Pro-Hyp regulates the Runx2 P1 promoter together with Foxg1. The present study revealed that Pro-Hyp is taken up by osteoblastic cells via the solute carrier family 15 member (Slc15a) 4. In the presence of Pro-Hyp, Runx2 is translocated from the nucleus to the cytoplasm and Foxg1 is translocated from the cytoplasm to the nucleus. We also found that Pro-Hyp promoted the interaction between Forkhead box o1 (Foxo1) and Runx2 and the dissociation of Foxg1 from Runx2. Moreover, we identified the Pro-Hyp response element in the Runx2 distal P1 promoter at nt -375 to -316, including the Runx2 binding sites and Fox core sequence. In the presence of Pro-Hyp, Runx2 is dissociated from the Pro-Hyp response element in the Runx2 distal P1 promoter. Subsequently, Foxg1 and Foxo1 activated the Runx2 promoter by binding to the Pro-Hyp response element. In summary, we delineated the mechanism by which Pro-Hyp stimulates the bone-related Runx2 distal P1 promoter activity in osteoblastic cells through Foxg1, Foxo1, and Runx2., 16p. Article No.BSR20210304 This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression., Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp

The inclusion complexations of paracyclophane (CP44) and β-cyclodextrin (β-CD) with several anilinonaphthalene sulfonic acids (ANSs) have been characterized via the enhancement of fluorescence spectra upon inclusion. β-CD and CP44 formed 1:1 inclusion complexes with ANSs, and the high stability of inclusion complexes of the latter was demonstrated. The thermodynamic parameters observed from the temperature dependence of the inclusion constants (van’t Hoff analysis) showed that CP44 inclusion complexations with ANSs are enthalpy-driven. Furthermore, the structure of the inclusion complexes was discussed based on 1H- and 2D ROESY-NMR measurements. It was found that CP44 encapsulates the naphthalene moiety of ANSs. On the other hand, differences in the structures of the β-CD inclusion complexes were observed for the 4-OH-substituted ANS. The molecular recognition for the inclusion of β-CD was found to be sensitive compared with that of CP44. In addition, the local polarity inside the CP44 and β-CD cavities was evaluated using ANSs as the fluorescence probe. Based on these results, we have suggested that the local polarity of ANSs in the hydrophobic cavities of CP44 and β-CD plays an important role in quantum yield enhancement upon inclusion.

A number of studies have shown that oral administration of collagen hydrolysate (CH) results in the absorption of di- and tri-peptides. In order to understand the dynamics of CH absorption and metabolism, molecular profiles of hydroxyproline (Hyp) and Hyp-containing peptides (HCPs) were analyzed by in situ perfusion of rat intestine and liver. The total amount of absorbed HCPs during 1 h of perfusion was 16.6 μmol, which was significantly higher than that of free Hyp (6.6 μmol). In addition, HCPs were also reliably detected in hepatic perfusate at the level higher than free Hyp. Thus, the results demonstrated that CH is absorbed predominantly as peptides, which subsequently enter systemic circulation. Size exclusion chromatography showed that perfusates include significant amount of HCPs larger than tripeptides, leading us to analyze these peptides in detail. Mass spectrometric analysis of intestinal perfusate finally identified three CH-derived peptides, which are surprisingly large as food-derived circulating peptides. Peptide quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that di- and tri-peptides, which are previously identified as major peptides in circulating blood, comprise only a part of HCPs in intestinal and liver perfusate. Finally, analysis of portal vein blood revealed that the larger peptides, such as pentadecapeptide identified in this study, could be absorbed in vivo. Taken all together, this study showed that peptides which are larger than tripeptide could reach to the circulation system after administration of CH, revealing previously unknown dynamics of absorption of CH.

A number of studies have shown that oral administration of collagen hydrolysate (CH) results in the absorption of di- and tri-peptides. In order to understand the dynamics of CH absorption and metabolism, molecular profiles of hydroxyproline (Hyp) and Hyp-containing peptides (HCPs) were analyzed by in situ perfusion of rat intestine and liver. The total amount of absorbed HCPs during 1 h of perfusion was 16.6 μmol, which was significantly higher than that of free Hyp (6.6 μmol). In addition, HCPs were also reliably detected in hepatic perfusate at the level higher than free Hyp. Thus, the results demonstrated that CH is absorbed predominantly as peptides, which subsequently enter systemic circulation. Size exclusion chromatography showed that perfusates include significant amount of HCPs larger than tripeptides, leading us to analyze these peptides in detail. Mass spectrometric analysis of intestinal perfusate finally identified three CH-derived peptides, which are surprisingly large as food-derived circulating peptides. Peptide quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that di- and tri-peptides, which are previously identified as major peptides in circulating blood, comprise only a part of HCPs in intestinal and liver perfusate. Finally, analysis of portal vein blood revealed that the larger peptides, such as pentadecapeptide identified in this study, could be absorbed in vivo. Taken all together, this study showed that peptides which are larger than tripeptide could reach to the circulation system after administration of CH, revealing previously unknown dynamics of absorption of CH.

The association constants (K) for the inclusion complexation of four kinds of cyclodextrins (CDs (β- and γ-), 2,6-di-O-methylated β-CD, and 2,3,6-tri-O-methylated β-CD) and cucurbit[7]uril (CB[7]) with 1,8- and 2,6-anilinonaphthalene sulfonic acids (ANSs) were determined from fluorescence spectra enhanced by inclusion. Various CDs and CB[7] form stable 1:1 inclusion complexes with 1,8- and 2,6-ANSs: K=80-11700 M(-1) for 2,6-ANS and 50-195 M(-1) for 1,8-ANS. The high stability of the inclusion complexes of 2,6-ANS with CB[7] and 2,6-di-O-methylated β-CD is shown. Further, we determined the fluorescence quantum yields (Φ values) for the inclusion complexes of ANSs by using a fluorescence spectrophotometer equipped with a half-moon unit. The Φ values of 1,8- and 2,6-ANSs were largely enhanced by the inclusion of methylated β-CDs and did not correlate with the degree of stability (K) of the inclusion complexes. We characterized the structures of the inclusion complexes by 2D ROESY-NMR measurements. In addition, the microenvironmental polarity inside the hydrophobic CD and CB[7] cavities was evaluated using the fluorescence probe 2,6-ANS. Based on the emission mechanism and the aspect of inclusion in a hydrophobic cavity, we have suggested that the microenvironmental polarity and viscosity for the excited state of ANS plays an important role for the Φ values of inclusion complexes.

An annual survey of the current national status of home parenteral nutrition (HPN) in Japan was begun in 1991, with a total of 231 registered patients from 142 institutions providing adequate data for evaluation and analysis. HPN was given for an average of 683±764 days to 93 patients with malignant diseases and 138 with benign disease, including 53 with inflammatory bowel disease and 79 with short bowel syndrome, 107 (46.3%) of whom were successfully rehabilitated. By the end of 1990, 61% of the patients investigated were still on HPN, 7% had already completed HPN, and 26% had died, the deceased accounting for 54% of the patients with malignant disease and 7% of those with benign diseases. A total of 321 catheters had been used by all 231 patients, the most common being the subcutaneously implanted type, accounting for 33% of all catheters. By the end of 1990, 32% of these 321 catheters were still in place, 18% had been removed on the termination of HPN and 44% had been removed due to complications of total parenteral nutrition, including 20% for catheter fever. Rehospitalization was required in 62% of the patients, the cause being HPN-related in 21% of the total patients. Metabolic complications were experienced by 60% of the patients, but none of these were severe although 6% required hospitalization. Thus, the total population of HPN patients and the success rate of rehabilitation in Japan were close to those reported in Europe, while the indications for HPN and its outcome were similar to those documented in the USA OASIS report, except that the incidence of rehospitalization from HPN-related causes in the Japanese survey was lower.

The rate of reaction between Leuco Bindschedler’s Green (BGH) and some p-benzoquinone derivatives (Q’s) in acetone was followed spectrophotometrically by means of the stopped-flow technique. The reaction was found to proceed reversibly. The rate data and the activation parameters for the forward and backward reactions were estimated. The rates were susceptible to the oxidation–reduction potentials of the Q’s. The absorption spectra observed at low temperatures indicated that three kinds of intermediates were involved. Radicals formed during the reaction were detected by the ESR method. The results have been discussed in relation to those previously obtained for systems involving Leuco Crystal Violet (LCV) and Leuco Malachite Green (LMG).

The effects of temperature and pressure on the formation of charge-transfer (CT) complexes between 2,6-dibromo-4-[(3,5-dibromo-4-hydroxyphenyl)(2-ethoxycarbonylphenyl)methylene]2,5-cyclohexadien-1-one (TBPE) and amines in nonpolar solvents have been studied, and the thermodynamic parameters estimated. It was found that the heat of formation (ΔH°) as well as the reaction volume (ΔV°) are largely negative, compared with those for ordinary CT complex formation. The electronic spectra in the visible region due to the outer CT complexes are well-characterized for primary and secondary amines in nonpolar solvents that have no proton-donating/-accepting nature. In other cases, the CT spectra are broad. Ionization of TBPE in polar solvents takes place as evidenced by the electronic spectrum of TBPE anion. However, ΔH° as well as ΔV° are unusually small. When Proton Sponge is used in nonpolar solvents, ion pairs are formed. In this case, ΔH° and ΔV° are largely negative as expected.

The Thermo-Plasticity of vitrains of five Japanese caking coals and four foreign caking coals have been examined from the ordinary A. S. T. M. test and some other fluidity measurements with the Gieseler plastmeter.The temperatures showing initial softening, fusion, maximum fluidity and solidification increases with rank of coal.The fluidity of Japanese coals is higher than that of foreign coals at about same rank. And the coal at about 86 percent carbon content has a maximum fluidity.This shows that the greater the content of tar and/or liquid-forming constituents in coals is.Around the initial softening point, the fluidity of coals hardly changes and shows an almost constant value for a long residence time at constant temperature.Around the fusion point, the fluidity of Miike coal (84.5%C) and Yubari coal (86.2%C) exceptionally increases with residence time up to about 150 minutes and then becomes almost constant up to 250 minutes. Flow curves for these coals at constant temperatures below the fusion points, obtained by measuring fluidities over a range of shear stress, indicate that these coals behave substantially as a plastic fluid.Above the fusion point, the fluidity of coals increases steeply at first, shows a maximum, and then decreases with increase of residence time at constant temperature.The time getting to the maximum fluidity increases with rank of coal at a constant temperature and decreases with increase of carbonizing temperature.

Much sample of uniformly heat-treated coal was prepared by using a nitrate bath in which the vessel containing a thin layer of coal sample was heated. The Japanese Nishikawachi strongly coking coal was added to the sixteen vitrain samples described in previous paper. These samples were heat treated with a heating rate of 3°C/ min. over the range from heat treatment temperature (HTT) 200°C to 500°C at intervals of 50°C.The yield of products the state of devolatilization have a strong correlation with the chemical constitution of original coals. Therefore, the yield of products at HTT 500°C is analogized from the proximate and ultimate analytical constitution of original coals.The analytical constitution of heat-treated coals depends on HTT and coal rank.In general, the beginning temperature of change in heat treatment increases with the increase of coal rank. The chemical constitution of heat-treated coals hardly changes below HTT about 350°C, except the lower rank coals below about 80% C. It seems, however, that the change of physical structure of heat-treated coals may happen below HTT about 350°C.At HTT about 400°C, the chemical constitution in coking coals of 83-87%C begins to change and the devolatilization rate of the lower rank coals shows a maximum. For coking coals, the devolatilization becomes violent and the ultimate analytical constitution of heat-treated coals changes largely at HTT about 450°C. At HTT about 500°C, the change of chemical constitution in strongly coking coals of about 90% C becomes remarkable.The reaction occured by heating up to HTT 500°C is mainly dehydration for lower rank coals. Dehydration and then demethanation predominate with the increase of coal rank.The aromaticity of heat-treated coals increases suddeny with the increase of HTT. The aromaticity and the chemical constitution of heat-treated coals at HTT 500°C do not show so large difference each other in comparison with the large difierence in original coals.

As part of a serial research work for coal structure, the IR spectra of the pyridine extracts of 16 different ranks of coal were measured by KBr method; from which some information in comparison with that of the original coals were obtained as follows.(1) Absorptions concerned with OH groupsThe absorption coefficients both of 3, 450 and 1, 260 cm-1, bands of the extract de-creased linearly, like that of the original coal, with the increase of the carbon content of the original coal.(2) Absorptions concerned with aliphatic CH hydrogensTwo peaks appeared at Taiheiya and Yubari, similar to the case of the original coal, on the plot of the absorption coefficient of 2, 920cm-1 band of the extract versus carbon content of the original coal, but rather higher coefficients were found in the extracts of lower-rank coals than in those original coal themselves. The absorption coefficient of 1, 450cm-1 band of the extract decreased as the carbon content of the original coal increased. While the absorption coefficient of 1, 380cm-1 band for the original coal had only one peak at Yubari, two peaks at Taiheiya and Yubari were found for that of the extract.(3) Absorptions concerned with Aromatic CH hydrogensIn stead of Bibai from which appeared the absorption at 3, 030cm-1 in case of the origi-nal coal, the band arose in the extract of a lower-rank coal, Takamatsu. In general, the absorption coefficient of 3, 030cm-1 band of the extract increased with increasing carbon content of the original coal except at Itmann, and had somewhat larger value than that of the corresponding coal. The absorption coefficients of 870, 820 and 750 cm-1 bands had quite similar trends with what were found in the original coal, though partial difference in the absorption coefficient of 750cm-1 band was noted.(4) The absorption band at 1, 600cm-1Whereas a lower coefficient compared with that of the original coal was seen for some lower rank coal extracts, no significant difference was found between the extract and the original coal for higher rank coals.The conclusion drawn from IR spectra observation was that the extracts obtained from the coals lower in rank than Bibai were richer in aliphatic structures and that the extratcs from higher rank coals had structures quite similar to their original coals. The deviation shown by Itmann coal extract was ascribed to the behavior of resinous material which might be extracted more from this sort of coal than to the behavior of the coal itself.

Some fundamental examinations were performed on the potassium bromide technique for the i. r. study of coal. The influences of grinding period and moisture content as well as applicability of the Lambert-Beer's law were examined and results were summarized as follows.1. When Yubari coal (86.2%C) was used as a sample, grinding the coal sample with potassium bromide for one hour was found to be sufficient to obtain nearly const-ant optical densities for all absorption bands except at 3450cm-1.2. The absorption band at 3450cm-1 assinged to hydroxyl group was apparently affected by moisture. Its optical density increased with time when a dried potassium bromide pellet was exposed to the air. The specific extinction coefficient, K (l/g·cm) of water was calculated to be 3. 58-3. 85 from the relation between the optical densities at 3450cm-4 and moisture content.3. The Lambert-Beer's law was confirmed to be applicable to the coal samples, and the specific extinction coefficients were caclulated from the linear relationship between coal concentrations and optical densities for almost all absorption bands.

石炭化度の異なる代表的試料(72.7～93.2%C)についての総合的研究の一環として,日本炭12炭種,外国炭5炭種につき,Wheeler法によってα,β,γ の3成分に分別し,各成分の抽出量ならびに蒸気圧平衡法による分子量を測定し,試料原炭の炭素含有量および脂肪族CH水素などとの関係について検討した。なおβ+γ 成分の抽出量,分子量について統計的解析法を適用した。β+γ 成分の抽出量,γ 成分の抽出量は石炭化度が増すにつれて増加し,86～87%Cの石炭で最高となり,さらに石炭化度が増加するにつれて急激な減少を示した。一般に日本炭の方が外国炭よりもβ+γ 成分の抽出量が多く,抽出量は統計的解析法によって求めた溶解度変数と関連することが認められた。β+γ 成分の分子量は460～1200程度であり,86～87%Cの石炭で最高となり,それ以後は石炭化度の増加につれて小さくなり,かつ統計的解析法によると,抽出物の単位構造当りの分子量は260～480程度の値を示した。γ成分の分子量は350～810程度であり,試料炭の脂肪族CH水素の多いほど分子量の大きいことがわかった。またβ 成分の分子量は,各成分間に加成性が成立つものとして推定値を算出した。

Six kinds of Japanese coals-Taiheiyo (77.8%C), Bibai (81.1%C), Akabira (83.4%C), Yubari (86.2%C), Hashima (87.2%C) and Nishikawachi (89. 8%C) were heat treated at the temperatures from 600 to 900°C and the yields of products, proximate and ultimate analyses including helium densities of the heat treated coals were investigated.

1. 夏期漬物に現われる亜硝酸の滔長を觀察した。2. キャベツ, 大根, 大根葉, 胡瓜, 茄子, 胃梅等の鹽漬, 糠味緡漬, 奈良漬, 味噌漬等何れも亜硝酸の存在を臉出し得るが, 日數の經つたもの, 酸味の張いものには存在しても痕跡程度である。3. 亜硝酸の現出は夏期 (15～23°の気温) には概ね漬込後3～5日目に最高に達するまうで此の點冬期 (3～5°) の14～21日目邊になるのに比べると頗る速い。4. 亜稍酸の最高價はグリース反應を使用して大根葉鹽漬の14日目に100cc中200mgを得た。之は清酒の生もと原酒母に現われる最高値の約200倍である。5. 同樣にしてキャベツの鹽漬では4日目4mg, 大根では4日目4.3mg, 胡瓜では3日目2mg, 恭子では4日目14.3mg, 青梅では2日目0.1mgの最高値を得た。6. ピックルには亜硝酸は認め得なかつた。之は酸性が強く亜硝酸は存在してもガスとして揮散する爲であろう。7. 他の一般の漬物に於ても酸が増加すれば亜硝酸は次第に淌失する。之は同じくガスとして揮散する爲である。8. キャベツ, 大根, 大根葉, 胡瓜, 茄子, 青梅中には多量の硝酸鹽 (汁液中0.04～0.47%) の存在をヂブェニラミソ硫酸比色法法で證明する事が出來るから之等を鹽漬にする時細菌の生育に俘い硝酸鹽が還元されて亜硝酸になるものである事が明かである。そして亜硝酸の最高量となるのは酸の生成しかかる頃である。9. 距稍酸鹽は血管壙大の作用があつて動脈硬化症, 狹心症等に亜礁酸アミル, 亜礁酸ソーダの如きが用いられる事がある事から考えれば其の常用量又は極量には及ばないが兎に角漬物中に現出する亜硝酸が之等の病氣の豫防に役立つ事は明である。從て本邦人の漬物好みも大いに意義がある事となる。本報告は松井が病中にあるのでたえず蓮絡を取りその方針に從つて試驗し取りまとめたものである。