ヒスチジンタグを持つホスファカンコア蛋白の大腸菌での発現と精製

Specific regions of core protein of phosphacan, one of the chondroitin sulfate proteoglycans, were expressed as fusion proteins with histidine-tag (His-tag) in Escherichia coli (E.coli) and were affinity purified using nickel-nitrilotriacetic acid (Ni-NTA) matrix. cDNA fragments encoding amino acid residues 343-446 (P3) and 1-340 (P4) of phosphacan core protein were amplified by polymerase chain reaction from E18 rat brain mRNA as template. The amplified products were subcloned into pQE30 vector and were introduced into E.coli strain M15 [pREP4] for the expression. The His-tagged fusion proteins were expressed by cultivating the transformants at 37℃ for 5h in the presence of 1mM IPTG. His-tagged P3 fusion protein (His-P3) was expressed as soluble form, and was purified using Ni-NTA matrix. His-tagged P4 fusion protein (His-P4) which was sequestered into insoluble inclusion bodies was treated with 8.0M urea to solubilize, and then was purified under denaturing conditions.コンドロイチン硫酸プロテオグリカンの一つであるホスファカンのコア蛋白の特定領域を,ヒスチジンタグ(His-tag)を持つ融合蛋白として大腸菌内で発現させニッケル-ニトリロ3酢酸(Ni-NTA)アフィニティ担体を用いて精製した｡ホスファカンコア蛋白のアミノ酸残基343-446(P3)及び1-340(P4)に相当するcDNA断片を,胎性18日目のラット脳由来のmRNAを鋳型としたPCRによって増幅した｡増幅された断片は発現ベクターpQE30に組み込まれ,これで大腸菌(M15[pREP4])を形質転換した｡His-tag融合蛋白の発現は形質転換株を1mM IPTG存在下で37℃,5時間培養することによって行われた｡His-tagged P3融合蛋白は可溶性蛋白質として発現し,Ni-NTA担体を用いて精製された.His-tagged P4融合蛋白は不溶性の封入体を形成したが,8M尿素によって可溶化され,変性条件下で同様に精製された

Base-induced Generation of Aryl(1,2,3-triazol-1-yl)carbenes from 1-[(N-Phenylsulfonyl)benzohydrazonoyl]-1,2,3-triazoles and Their Ring Enlargement to 3-Aryl-1,2,4-triazines

The Bamford-Stevens reactions of 1-[N-(phenylsulfonyl)benzohydrazonoyl]-1,2,3-triazole derivatives, readily available from the 1,3-dipolar cycloadditions of N-(phenylsulfonyl)benzohydrazonoyl azides with enamines, were examined and the transformation from aryl(1,2,3-triazol-1 -yl)carbene intermediates to the corresponding 3-aryl-1,2,4-triazine derivatives was first observed.ArticleHETEROCYCLES. 65(2): 279-286 (2005).journal articl

Possible role of adenosine in macula densa control of glomerular hemodynamics

Possible role of adenosine in macula densa control of glomerular hemodynamics.BackgroundThe macula densa (MD), a plaque of specialized tubular epithelial cells, senses changes in tubular NaCl concentration and sends a signal(s) that controls the resistance of the glomerular afferent arteriole (Af-Art). This mechanism, called tubuloglomerular feedback (TGF), is thought to be important in the homeostasis of body fluids and electrolytes. Our aim was to determine the range of NaCl concentrations in tubular fluid at the MD that would elicit the Af-Art response. In addition, we examined the possible involvement of adenosine in transmitting the signal from the MD to the Af-Art.MethodsRabbit Af-Arts and attached MD were simultaneously microperfused in vitro, keeping pressure in the Af-Art at 60mm Hg.ResultsIncreasing the Na+/Cl- concentration of the MD perfusate from 26/7 to 41/22 mEq/L decreased the luminal diameter of the terminal Af-Art segment by 10 ± 4% (N = 9; P < 0.01). The response was maximal at 55/36 mEq/L (18 ± 6%), so that further elevation of NaCl concentration had no additional effect (20 ± 6% at 84/65 mEq/L). When FK838 (10-6 mol/L), a specific adenosine A1 receptor antagonist, was added to both Af-Art perfusate and bath, Af-Art constriction was completely abolished. The maximum response was 20 ± 3% before FK838 and 0.6 ± 1% afterward (N = 12). Adding adenosine at 10-8 mol/L to both bath and perfusate significantly augmented Af-Art constriction induced by increased NaCl at the MD (P < 0.01); however, adding 10-8 to 10-6 mol/L adenosine to the MD perfusate had no effect regardless of the NaCl concentration at the MD.ConclusionsThese results demonstrate that MD control of Af-Art resistance is induced by relatively low NaCl concentrations at the MD, and that activation of the adenosine A1 receptor in the vascular and interstitial space (but not the tubular lumen) may be essential for signal transmission from the MD to the Af-Art

Atomic-scale dents on cellulose nanofibers: the origin of diverse defects in sustainable fibrillar materials

セルロースナノファイバーに生じた原子レベルの欠陥構造を発見 --理想的なバイオポリマー材料の生産にむけて--. 京都大学プレスリリース. 2022-10-07.Atomic-scale dent structures on the surfaces of cellulose nanofibers were detected. These dent parts constituted at least 30–40% of the total length of the dispersed nanofibers, and deep dents induced the kinking and fragmentation of nanofibers

Optical-power-dependent splitting of magnetic resonance in nitrogen-vacancy centers in diamond

Nitrogen-vacancy (NV) centers in diamonds are a powerful tool for accurate magnetic field measurements. The key is precisely estimating the field-dependent splitting width of the optically detected magnetic resonance (ODMR) spectra of the NV centers. In this study, we investigate the optical power dependence of the ODMR spectra using NV ensemble in nanodiamonds (NDs) and a single-crystal bulk diamond. We find that the splitting width exponentially decays and is saturated as the optical power increases. Comparison between NDs and a bulk sample shows that while the decay amplitude is sample-dependent, the optical power at which the decay saturates is almost sample-independent. We propose that this unexpected phenomenon is an intrinsic property of the NV center due to non-axisymmetry deformation or impurities. Our finding indicates that diamonds with less deformation are advantageous for accurate magnetic field measurements.Comment: 9 pages, 7 figure

ヒト血清リポタンパク質とシクロデキストリンのアガロースゲル内における相互作用

Cyclodextrins (CDs) are cyclic oligosaccharides with low molecular weight. They have been known to bind to some serum lipoproteins and to form complexes. To elucidate whether each serum lipoprotein subclass could be separated by electrophoresis using CDs, we performed preliminary experiment, in which lipoprotein-CDs interaction was examined on electrophoresis with agarose gel. When the supporting agarose gel containing both α-CD and β-CD was prepared and was applied to isoelectric focusing for fractionating serum lipoproteins, apoB lipoproteins were found to be clearly separated into several fractions on this electrophoresis. This finding suggested that apoB lipoprotein may be detected as isolated form by arranging amounts of added CDs.シクロデキストリンは低分子量の環状オリゴ糖で, リポタンパク質と複合体を形成する｡シクロデキストリンを使用した電気泳動による血清リポタンパク質サブクラスの分離の可能性を明らかにするために, リポタンパク質とシクロデキストリンとの泳動用ゲル内での相互作用について検討をおこなった｡その結果,α-CDとβ-CDの2種類のCDをアガロースゲルに添加し,このゲルを泳動用支持体として等電点電気泳動をおこなったところ,apoB含有リポタンパク質が数分画分離された｡したがって,ゲル内へのシクロデキストリンの適正な添加条件が設定できるならば,apoB含有リポタンパク質サブクラスの分離が可能となることが示唆された

Photopolarimetric Monitoring of Blazars in the Optical and Near-Infrared Bands with the Kanata Telescope. I. Correlations between Flux, Color, and Polarization

We report on the correlation between the flux, color and polarization variations on time scales of days--months in blazars, and discuss their universal aspects. We performed monitoring of 42 blazars in the optical and near-infrared bands from 2008 to 2010 using TRISPEC attached to the "Kanata" 1.5-m telescope. We found that 28 blazars exhibited "bluer-when-brighter" trends in their whole or a part of time-series data sets. This corresponds to 88% of objects that were observed for >10 days. Thus, our observation unambiguously confirmed that the "bluer-when-brighter" trend is common in the emission from blazar jets. This trend was apparently generated by a variation component with a constant and relatively blue color and an underlying red component. Prominent short-term flares on time scales of days--weeks tended to exhibit a spectral hysteresis; their rising phases were bluer than their decay phases around the flare maxima. In contrast to the strong flux--color correlation, the correlation of the flux and polarization degree was relatively weak; only 10 objects showed significant positive correlations. Rotations of polarization were detected only in three objects: PKS 1510-089, 3C 454.3, and PKS 1749+096, and possibly in S5 0716+714. We also investigated the dependence of the degree of variability on the luminosity and the synchrotron peak frequency, \nu_peak. As a result, we found that lower luminosity and higher \nu_peak objects had smaller variations in their amplitudes both in the flux, color, and polarization degree. Our observation suggests the presence of several distinct emitting sources, which have different variation time-scales, colors, and polarizations. We propose that the energy injection by, for example, internal shocks in relativistic shells is a major factor for blazar variations on time scales of both days and months.Comment: 39 pages, accepted for publication in PAS

Admissible Sectional Dimensions of R/C Floor Elements to be Designed without Deflection Check Part 2: Floor Slabs

We review the adequacy of the slab thickness equation in the r/c design code by the Architectural Institute of Japan, in comparison with the related data obtained by our recently renovated method which employs the known effective stiffness and an experimentally estimated time-dependent reduced elastic modules and whose practical accuracy to tolerable degrees has been observed in our earlier reports. Using this method we try series of deflection estimate for slab models of graduated dimensions in a practically chosen wide range, for the purpose of examining the subject matter of defined possibility for rational slab sections to be designed without deflection check