Enzymatic Decomposition of Basic Amino Acids by the Cell Free Extract of a Red Alga， Gymnogongrus flabelliformis

紅藻の一種オキツノリGymnogongrus flabelliformisを-200℃に凍結貯蔵すると藻体遊離アミノ酸の主要成分であるgigartinineおよびcitrullineが著しく減少し7週間の貯蔵で両者とも95%以上が分解された.藻体をpH8.0のTris緩衝液とともに磨砕して得た抽出液を25℃に置いた場合には更に顕著で、あって，gigartinineは3時間で完全に消失した.更に透析処理した抽出液に17種のアミノ酸混合液を添加して分解性をみたところ，この抽出液は特に塩基性アミノ酸を強く分解することを知った. 以上のことは本海藻に塩基性アミノ酸を特異的に分解する酵素の存在を示唆したので，この反応に対する緩衝液の種類の影響，至適温度，pH，基質特異性，分解生産物について調べた.結果は以下の如くである. (1) 燐酸緩衝液はTris緩衝液.硼砂緩衝液に比較してarginine分解の活性を低下させる. (2) 反応の至適温度は30℃附近にある. (3) 反応の至適pHは8.0～9.0の間にある. (4) 塩基性アミノ酸であるL-arginine ，L-ornithine， L-lysine，L-gigartinineは強く分解するが，L-histidine L-methionineにも弱い分解力を持つ.DL-Ornithine，DL-lysine，DL-gigartinineは50%しか分解されず，この反応はL-アミノ酸に特異的であると推察される. (5) Arginine，gigartinineの分解生産物をペーパークロマトグラフィーによって検索したところ，いずれも主生成物として相当するα-ケト酸が検出された.したがって，この海藻抽出液には塩基性アミノ酸の酸化的脱アミノ反応に関与する酵素の存在が推定された.The red alga, Gymnogongrus flabelliformis has a peculiar free amino acid pattern, in which one characteristic amino acid, gigartinine, is the most dominant component1-4). Recently, we observed that the contents of gigartinine and citrulline in the extracts of the alga decreased markedly during storage at about -20℃. The purpose of this paper is to report that the cell free extract of Gymnogongrus flabelliformis has an enzymatic activity for the decomposition of basic L-amino acids. 1) The cell free extract was prepared by grinding the alga with an equal volume of pH 8.0 Tris buffer in a mortar previously chilled and centrifuging for 30 min. at 10,000 rpm under chilling. The supernatant obtained was dialyzed against distilled water for 24 hours. 2) The extract showed a deaminative activity towards L-gigartinine, L-arginine, L-lysine and L-ornithine, and decomposed 50% of DL-gigartinine, DL-lysine and DL-ornithine. It suggests that D-isomers are not affected. 3) The influence of the different kinds of buffer solution on the decomposing activity of the extract towards arginine was examined. The phosphate buffer showed a slightly inhibitive effect. 4) Effects of pH and temperature on the decomposing activity of the extract were examined. The pH optimum was relatively broad. The activity of the extract did not vary appreciably from pH 8.0 to 9.0. The optimum temperature was about 30℃. 5) The decomposition products of arginine and gigartinine were surveyed by paperchromatography and the corresponding a-keto acid to each one of the amino acids was detected, respectively. The data suggest that the extract of the alga has an activity to catalyze oxidative deamination of basic L-amino acids

周波数変調原子間力顕微鏡を用いたサブナノスケール３次元水和搖動構造解析

13301甲第4829号博士（工学）金沢大学博士論文要旨Abstract 要約Outlin

数種緑藻の遊離アミノ酸とペプチッド

7種の海産緑藻のエキスの遊離アミノ酸組成を調べた。その結果,アナアオサ(Ulva pertusa)にジペプチッド,L-arginyl-L-glutamineが著量に存在することを認めた。このペプチッドはイオン交換樹脂カラムクロマトグラフィーにより単離され,加水分解生成物の同定および合成によりその構造が確認された。アナアオサではL-arginyl-L-glutamineは全エキス窒素の約20%を占め,主成分をなしていた。さらにウスバアオノリ(Enteromorpha linza)にも検出されたが,外の5種には認められなかった。ウスバアオノリではarginyl-glutamineとともにprolineの含量が比較的高かった。この外,アサミドリシオグサ(Cladophora densa)ではglycineとproline,マユハキモ(Chlorodesmis comosa),ハイミル(Codium adhaerens)およびミル(Codium fragile)ではglutamic acidとglutamine,スリコギイワヅタ(Caulerpa racemosa)ではglycineの含量がそれぞれ高かった。 またこれら緑藻におけるアミノスルフォン酸の分布をペーパークロマトグラフィーにより調べた。その結果,taurineを4種の海藻に,D-cysteinolic acidを3種に,N-monomethyltaurineを2種に,homotaurineを1種にそれぞれ検出した。このうちアサミドリシオグサではhomotaurineを分離して確認した。The amino acid compositions of 7 marine green algal extracts were examined by means of an amino acid analyzer. Ulva pertusa was for the first time found to contain a dipeptide, L-arginyl-L-glutamine, in a large quantity. This peptide was detected also in Enteromorpha linza but not in the other five species. Glutamic acid and glutamine were relatively predominant in Codium fragile, C. adhaerens and Chlorodesmis comosa. In Caulerpa racemosa, the level of glycine was remarkably high. Glycine and proline were predominant in Cladophora densa. Aminosulfonic acids in these algae were examined by paper chromatography. Taurine was detected in 4 species, D-cysteinolic acid in 3, N-monomethyltaurine in 2, and homotaurine in 1, respectively. Occurrence of homotaurine in Cladophora densa was also established

7種海藻中における第4級アンモニウム塩基の分布

最近,海洋生化学資源の開発が重要視され,海産物由来のいくつかの有効物質が単離・同定されて,その生化学的および薬理学的役割が検討されてきている。その中で,海藻中にも2,3の有効物質が明らかにされてはいるものの,その数は少ない。本研究では,海藻中の有効物質が含窒素化合物に多いことから,海藻中の第4級アンモニウム塩基を7種の紅藻および緑藻を用いて検索した。 各海藻の70%エタノール抽出液中の第4級アンモニウム塩基をイオン交換カラムクロマトグラフィーを用いて単離・精製し,そのTLC上のRf値,IRスペクトルおよび融点を測定して,購入および合成した既知の第4級アンモニウム塩基のそれらと比較することにより同定した。その結果, laminine, candicine, stachydrine, glycine betaine, betonicine, β-homobetaineおよびγ-boutyrobetaineと2種の未同定塩基を検出した。candicineが海藻中から単離・同定されたのは,今回が最初と思われる。これら塩基の海藻種間における分布に関しては,特に特異性は認められなかった。Seven species of marine red and green algae were examined for quaternary ammonium bases. Nine bases were purified and isolated by ion exchange column chromatography from the ethanolic extracts of the algae. The isolated bases were identified by measuring their Rf values in thin layer chromatography, infrared spectra and melting points in comparison with specimen of thirteen kinds of authentic quaternary ammonium bases which were prepared by commercial means or syntheses. As the results, seven bases; laminine, candicine, stachydrine, glycine betaine, betonicine, β-homobetaine and γ-butyrobetaine, and two unidentified bases were detected. It was first demonstrated that candicine is present in marine algae. There is no essential difference in the distribution of the bases among the species of marine algae examined here

Epidemiological features of acute medial meniscus posterior root tears

Purpose　Untreated or overlooked medial meniscus posterior root tears (MMPRTs) induce sequential knee joint degradation. We evaluated epidemiological features of acute MMPRT for its early detection and accurate diagnosis. Methods　Among 330 MMPRT patients from 2018 to 2020, those who underwent arthroscopic pullout repairs were enrolled. Patients who underwent non-operative treatment or knee arthroplasty, those with a cruciate ligament-deficient knee or advanced osteoarthritis of the knee, and those with insufficient data were excluded. Finally, we retrospectively evaluated data from 234 MMPRTs (female: 79.9%, complete tears: 92.7%, mean age: 65 years). Welch’s t-test and Chi-squared test were used for pairwise comparisons. Spearman’s rank correlation analysis was performed between age at surgery and body mass index (BMI). Multivariable logistic regression analysis with stepwise backward elimination was applied to the values as risk factors for painful popping events. Results　In both sexes, there were significant differences in height, weight, and BMI. In all patients, there was a significant negative correlation between BMI and age (ρ =  − 0.36, p  Conclusion　Higher BMI was associated with a significantly younger age of MMPRT onset. Partial MMPRTs had a low frequency of painful popping events (43.8%)

Tip dependence of three-dimensional scanning force microscopy images of calcite-water interfaces investigated by simulation and experiments

In this study, we have investigated the influence of the tip on the three-dimensional scanning force microscopy (3D-SFM) images of calcite-water interfaces by experiments and simulations. We calculated 3D force images by simulations with the solvent tip approximation (STA), Ca, CO(3)and OH tip models. For all the 3D images, thezprofiles at the surface Ca and CO(3)sites alternately show oscillatory peaks corresponding to the hydration layers. However, the peak heights and spacings become larger when the mechanical stability of the tip becomes higher. For analyzing thexyslices of the 3D force images, we developed the extended STA (E-STA) model which allowed us to reveal the strong correlation between the hydration structure just under the tip and the atomic-scale force contrasts. Based on these understandings on the image features showing the strong tip dependence, we developed a method for objectively estimating the similarity between 3D force images. With this method, we compared the simulated images with the three experimentally obtained ones. Among them, two images showed a relatively high similarity with the image obtained by the simulation with the Ca or the CO(3)tip model. Based on these agreements, we characterized the hydration structure and mechanical stability of the experimentally used tips. The understanding and methodology presented here should help us to derive accurate information on the tip and the interfacial structure from experimentally obtained 3D-SFM images.Peer reviewe

Tubulointerstitial nephritis complicating IVIG therapy for X-linked agammaglobulinemia

BACKGROUND: Patients with X-linked agammaglobulinemia (XLA) develop immune-complex induced diseases such as nephropathy only rarely, presumably because their immunoglobulin (Ig) G concentration is low. We encountered a patient with XLA who developed tubulointerstitial nephritis during treatment with intravenous immunoglobulin (IVIG). CASE PRESENTATION: A 20-year-old man was diagnosed with XLA 3 months after birth and subsequently received periodic γ-globulin replacement therapy. Renal dysfunction developed at 19 years of age in association with high urinary β2-microglobulin (MG) concentrations. A renal biopsy specimen showed dense CD3-positive lymphocytic infiltration in the tubulointerstitium and tubular atrophy, while no IgG4-bearing cell infiltration was found. Fibrosclerosis and crescent formation were evident in some glomeruli. Fluorescent antibody staining demonstrated deposition of IgG and complement component C3 in tubular basement membranes. After pulse steroid therapy was initiated, urinary β2-MG and serum creatinine concentrations improved. CONCLUSION: Neither drug reactions nor collagen disease were likely causes of tubular interstitial disorder in this patient. Although BK virus was ruled out, IgG in the γ-globulin preparation might have reacted with a pathogen present in the patient to form low-molecular-weight immune complexes that were deposited in the tubular basement membrane

Improvements in fundamental performance of liquid-environment atomic force microscopy with true atomic resolution

Recently, there have been significant advancements in liquid-environment atomic force microscopy (AFM) with true atomic resolution. The technical advancements are followed by a rapid expansion of its application area. Examples include subnanometer-scale imaging of biological systems and three-dimensional measurements of water distributions (i.e., hydration structures) and fluctuating surface structures. However, to continue this progress, we should improve the fundamental performance of liquid-environment dynamic-mode AFM. The present AFM technique does not allow real-time imaging of atomic-scale dynamic phenomena at a solid-liquid interface. This has hindered atomic-level understanding of crystal growth and dissolution, catalytic reactions and metal corrosion processes. Improvement in force sensitivity is required not only for such a high-speed imaging but also for various surface property measurements using a high-resolution AFM technique. In this review, we summarize recent works on the improvements in the force sensitivity and operation speed of atomic-resolution dynamic-mode AFM for liquid-environment applications. © 2015 The Japan Society of Applied Physics.Embargo Period 12 month