シロイヌナズナ由来過酸化リン脂質グルタチオンペルオキシダーゼ様遺伝子のクローニングと発現

A cDNA encoding Arabidopsis purative phosphplipid hydroperoxide gultathione peroxidase (PHGPX) was cloned and sequenced by the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. The cDNA comprised 803 bp, and included an open reading frame which encodes a polypeptide of 169 amino acid residues with a molecular mass of 18,600 Da. The deduced amino acid sequence showed homology to plant putative PHGPXs and mammalian PHGPXs. The cloned gene was expressed in Escherichia coli cells to prouce an extra protein, which showed a molecular mass similar to the deduced one.シロイヌナズナから過酸化リン脂質グルタチオンペルオキシダーゼと高い相同性を持つタンパク質をコードするcDNAを単離し、その塩基配列を決定した。本遺伝子は、全長803bpからなり、169アミノ酸残基のタンパク質をコードしていた。アミノ酸配列は植物由来過酸化リン脂質グルタチオンペルオキシダーゼ様タンパク質と約80％の相同性を、哺乳類由来過酸化リン脂質グルタチオンペルオキシダーゼと約50％の相同性を示した。本遺伝子を大腸菌中で発現させた結果、遺伝子から予測される分子量をもつタンパク質が新たに生産された

ホウレンソウ種子に存在するα-グルコシダーゼの分子多型変化

Two molecular forms of α-glucosidase were isolated from spinach seeds after storage at 4℃ by CM-cellulose column chromatography and gel filtration. The molecular masses of α-glucosidase A and B were 78 kDa and 82 kDa by SDS-PAGE, and 62 kDa and 70 kDa by gel filtration, respectively. α-Glucosidase A had high activity not only toward maltooligosaccharides but also toward α-glucans. The optimum pH was 4.5-5.5 and about 50% of the activity remained after incubation at 65℃ for 20 min. On the other hand, α-glucosidase B had high activity toward maltooligosaccharides but faint activity toward α-glucans. The optimum pH was 5.0 and no activity was found after incubation at 65℃ for 20 min. The enzymatic and immunological properties of α-glucosidase A and B were similar to those of α-glucosidase. Ⅰor Ⅱ, and α-glucosidase Ⅲ or Ⅳ, isolated from spinach seeds without 4℃ storage, respectively. These findings suggest that the α-glucosidase in spinach seeds is modified to be two molecular forms.4℃保存したホウレンソウ種子から2種のα-グリコシダーゼをCM-セルロースカラムクロマトグラフィーとゲル濾過により精製した。α-グルコシダーゼAとBの分子量はそれぞれSDS-PAGEで78kDA、82kDa、ゲル濾過で62kDa、70kDaであった。α-グルコシダーゼAはマルトオリゴ糖だけではなく可溶性デンプンに対して強い加水分解活性を示した。至適pHは4.5-5.5であり、65℃、20分処理後に約50％の残存活性を示した。一方、α-グルコシダーゼBはマルトオリゴ等に対して強い加水分解活性を示したが、可溶性デンプンに対してほとんど活性を示さなかった。至適pHは5.0であり、65℃、20分処理後に活性を消失した。α-グルコシダーゼAとα-グルコシダーゼBは、4℃保存を行わない種子に見出されたα-グルコシダーゼⅠ、Ⅱとα-グルコシダーゼⅢ、Ⅳにそれぞれ酵素化学的性質、免疫化学的性質が類似していた。これらの結果、ホウレンソウ種子に存在するα-グルコシダーゼの分子多型は2種類に収束されることが示唆された

Rice Nudix Hydrolase OsNUDX2 Sanitizes Oxidized Nucleotides

Nudix hydrolase (NUDX) hydrolyzes 8-oxo-(d)GTP to reduce the levels of oxidized nucleotides in the cells. 8-oxo-(d)GTP produced by reactive oxygen species (ROS) is incorporated into DNA/RNA and mispaired with adenine, causing replicational and transcriptional errors. Here, we identified a rice OsNUDX2 gene, whose expression level was increased 15-fold under UV-C irradiation. The open reading frame of the OsNUDX2 gene, which encodes 776 amino acid residues, was cloned into Escherichia coli cells to produce the protein of 100 kDa. The recombinant protein hydrolyzed 8-oxo-dGTP, in addition to dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), as did Arabidopsis AtNUDX1; whereas the amino acid sequence of OsNUDX2 had 18% identity with AtNUDX1. OsNUDX2 had 14% identity with barley HvNUDX12, which hydrolyzes 8-oxo-dGTP and diadenosine tetraphosphates. Suppression of the lacZ amber mutation caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree when the OsNUDX2 gene was expressed in mutT-deficient E. coli cells. These results suggest that the different substrate specificity and identity among plant 8-oxo-dGTP-hydrolyzing NUDXs and OsNUDX2 reduces UV stress by sanitizing the oxidized nucleotides

Serine racemases from barley, Hordeum vulgare L., and other plant species represent a distinct eukaryotic group: gene cloning and recombinant protein characterization

Several D-amino acids have been identified in plants. However, the biosynthetic pathway to them is unclear. In this study, we cloned and sequenced a cDNA encoding a serine racemase from barley which contained an open reading frame encoding 337 amino acid residues. The deduced amino acid sequence showed significant identity to plant and mammalian serine racemases and contained conserved pyridoxal 5-phosphate (PLP)-binding lysine and PLP–interacting amino acid residues. The purified gene product catalyzed not only racemization of serine but also dehydration of serine to pyruvate. The enzyme requires PLP and divalent cations such as Ca2+, Mg2+, or Mn2+, but not ATP, whereas mammalian serine racemase activity is increased by ATP. In addition to the results regarding the effect of ATP on enzyme activity and the phylogenetic analysis of eukaryotic serine racemases, the antiserum against Arabidopsis serine racemase did not form a precipitate with barley and rice serine racemases. This suggests that plant serine racemases represent a distinct group in the eukaryotic serine racemase family and can be clustered into monocot and dicot types.</p

外反母祉における種子骨の偏位と関節症変化の関係

BACKGROUND: Although the tangential sesamoid view is used to visualize the sesamoid position relative to the first metatarsal head, correctly evaluating patients with severe varus of the first metatarsal is difficult. Computed tomography (CT) can be helpful due to its cross-sectional images in any plane. The purposes of this study were to evaluate the alignment of the tibial sesamoid and investigate the relationship between malalignment and degenerative change in the sesamoid metatarsal joint (SMJ) using simulated weight-bearing CT imaging in patients with hallux valgus. METHODS: In total, 269 feet from 142 patients with hallux valgus were included. The mean age was 63.7 years (range, 33-87 years). An anteroposterior weight-bearing radiograph was assessed for sesamoid position into 3 grades: grade 1, the tibial sesamoid was medial to the axis of the first metatarsal; grade 2, the tibial sesamoid was located below the first metatarsal axis; and grade 3, the tibial sesamoid was lateral to the first metatarsal axis. The hallux valgus and intermetatarsal angles (HVA and IMA, respectively) were measured. The lateral shift of the tibial sesamoid relative to the first metatarsal was classified into 3 grades on simulated weight-bearing CT classification: grade 1, tibial sesamoid was entirely medial to the intersesamoid ridge; grade 2, tibial sesamoid was subluxated laterally but located below the intersesamoid ridge; and grade 3, tibial sesamoid was located entirely lateral to the intersesamoid ridge. The differences of HVA and IMA in each grade were confirmed by using 1-way analysis of variance with Bonferroni post hoc corrections. Furthermore, multiple linear regression analysis was used to predict the degenerative change in the SMJ for age, sex, sesamoid position determined by CT or plain radiography, HVA, and IMA. The χ2 test was used for descriptive statistics to analyze the agreement between radiography or CT classifications of sesamoid position against degenerative change in the SMJ. RESULTS: Based on the radiographic classification of the tibial sesamoid position, 7 feet were classified as grade 1, 72 were grade 2, and 190 were grade 3, respectively. Based on the CT classification, 34 feet were classified as grade 1, 116 were grade 2, and 119 were grade 3. Degenerative change in SMJ progressed according to the sesamoid shift relative to the first metatarsal using either radiography or CT. In radiography, statistically significant differences were found except for the difference in HVA between grades 1 and 2. In addition, statistically significant differences were found between HVA and IMA, along with the grades in CT.In multiple linear regression, degenerative change was correlated with age and sesamoid position in CT and radiographic classifications. CONCLUSION: Our study showed that lateral shift of the tibial sesamoid increased in association with progression of the hallux valgus deformity. Furthermore, increasing lateral shift of the tibial sesamoid was associated with worsening degenerative change within the SMJ. LEVEL OF EVIDENCE: Level III, retrospective comparative study.博士（医学）・甲第657号・平成28年11月24日© Copyright 2016 American Orthopaedic Foot & Ankle SocietyThe definitive version is available at " http://dx.doi.org/10.1177/1071100716661827

ケイ酸/亜鉛置換ストロンチウムアパタイトコーティングによるβ-リン酸カルシウムの骨誘導能促進効果

Background: β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties. Methods: The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP). Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks. In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically. Results: Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group. Conclusions: Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.博士（医学）・甲第805号・令和3年12月21日© 2021. The Author(s).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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data

Timolol activates the enzyme activities of human carbonic anhydrase I and II.

Timolol, a beta-blocker, has been shown to be an effective ocular hypotensive agent when used alone or with carbonic anhydrase inhibitor on ocular hypertensive or open angle glaucoma patients. The effect of timolol hemihydrate on the CO(2) hydration activities of human carbonic anhydrase (HCA) I and II and their reaction mechanisms were investigated. Timolol activates the enzyme activities of HCA I and HCA II. In HCA I and II, the enzyme kinetic results clearly showed that timolol increases the value of V(max) but does not influence the value of K(m). The enzyme kinetic method showed that timolol noncompetitively activates HCA I and II activities through the formation of a ternary complex consisting of the enzyme, the substrate, and timolol. These results indicate that timolol binds apart from the narrow cavity of the active site. AutoDocking results showed that timolol binds at the entrance of the active site cavity in a region where the proton shuttle residue, His 64, of HCA I or II, is placed. The enzyme kinetic and AutoDocking results showed that timolol might weakly bind near the proton shuttle residue, His 64, to accelerate the proton transfer rate from His 64 to the buffer components. It is known that efficient activators of carbonic anhydrase possess a bulky aromatic/heterocyclic moiety and a primary/secondary amino group in their molecular structure. Timolol has a heterocyclic moiety and a secondary amino group, which are typical structures in efficient activators of carbonic anhydrase.Timolol, a beta-blocker, has been shown to be an effective ocular hypotensive agent when used alone or with carbonic anhydrase inhibitor on ocular hypertensive or open angle glaucoma patients. The effect of timolol hemihydrate on the CO(2) hydration activities of human carbonic anhydrase (HCA) I and II and their reaction mechanisms were investigated. Timolol activates the enzyme activities of HCA I and HCA II. In HCA I and II, the enzyme kinetic results clearly showed that timolol increases the value of V(max) but does not influence the value of K(m). The enzyme kinetic method showed that timolol noncompetitively activates HCA I and II activities through the formation of a ternary complex consisting of the enzyme, the substrate, and timolol. These results indicate that timolol binds apart from the narrow cavity of the active site. AutoDocking results showed that timolol binds at the entrance of the active site cavity in a region where the proton shuttle residue, His 64, of HCA I or II, is placed. The enzyme kinetic and AutoDocking results showed that timolol might weakly bind near the proton shuttle residue, His 64, to accelerate the proton transfer rate from His 64 to the buffer components. It is known that efficient activators of carbonic anhydrase possess a bulky aromatic/heterocyclic moiety and a primary/secondary amino group in their molecular structure. Timolol has a heterocyclic moiety and a secondary amino group, which are typical structures in efficient activators of carbonic anhydrase

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception

植物間コミュニケーションの仕組みを解明 --受容した香りを防御物質に変える遺伝子発見--. 京都大学プレスリリース. 2023-02-28.Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl β-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl β-D-glucopyranoside arabinosylation to produce HexVic in planta