Renaturation of the mature subtilisin BPN' immobilized on agarose beads

AbstractWe report here another example of renaturation of subtilisin BPN′(Sbtl) by using an immobilized preparation instead of applying a digestible mutant of Streptomyces subtilisin inhibitor (SSI), a proteinaceous inhibitor of Sbtl [M. Matsubara et al. (1994) FEBS Letters 342, 193–196]. The mature Sbtl was immobilized on agarose beads employing the amino group of the protein. After thorough washing, the immobilized Sbtl was subjected to denaturation in 6 M guanidine hydrochloride (GdnHCl) at pH 2.4 for 4 h, followed by renaturation in 2 M potassium acetate at pH 6.5 for 24 h. This denaturation/renaturation cycle was repeated five times. The recovered activity of the renatured immobilized Sbtl settled at a constant level after the third denaturation/renaturation cycle, demonstrating that almost 100% renaturation was attained by use of the immobilized Sbtl. This immobilized Sbtl preparation could well be utilized for the mechanistic study of protein folding. We then found that 2 M potassium acetate was superior to 2 M potassium chloride as a refolding medium and that the ability of SSI to induce the correct shape of the mature Sbtl was lacking in several refolding media in both thermodynamic and kinetic criteria. Thus the main cause for the increase of refolding yield of Sbtl by coexistence of SSI was prevention of the autolysis of Sbtl

Photoinduced 1,2-Hydro(cyanomethylation) of Alkenes with a Cyanomethylphosphonium Ylide

An efficient method has been developed for the 1, 2-hydro(cyanomethylation) of alkenes, in which a cyanomethyl radical species is generated from a cyanomethylphosphonium ylide by irradiation with visible light in the presence of an iridium complex, a thiol, and ascorbic acid. The cyanomethyl radical species then adds across the C=C double bond of an alkene to form an elongated alkyl radical species that accepts a hydrogen atom from the thiol to produce an elongated aliphatic nitrile. The ascorbic acid acts as the reductant to complete the catalytic cycle

The newt (Cynops pyrrhogaster ) RPE65 promoter: molecular cloning, characterization and functional analysis

The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.8 kb RPE65 promoter from the newt, Cynops pyrrhogaster. Sequence analysis revealed several conserved regulatory elements described previously in mouse and human RPE65 promoters. Second, having previously established an I-SceI-mediated transgenic protocol for the newt, we used it here to examine the −657 bp proximal promoter of RPE65. The promoter assay used with F0 transgenic newts confirmed transgene expression of mCherry fluorescent protein in the RPE. Using bioinformatic tools and the TRANSFAC database, we identified a 340 bp CpG island located between −635 and −296 bp in the promoter; this region contains response elements for the microphthalmia-associated transcription factor known as MITF (CACGTG, CATGTG), and E-boxes (CANNTG). Sex-determining region box 9 (or SOX9) response element previously reported in the regulation of RPE genes (including RPE65) was also identified in the newt RPE65 promoter. Third, we identified DNA motif boxes in the newt RPE65 promoter that are conserved among other vertebrates. The newt RPE65 promoter is an invaluable tool for site-specific delivery of exogenous genes or genetic manipulation systems for the study of retinal regeneration in this animal

Impact of noncontrast PCI for ACS

Purpose : Contrast-induced acute kidney injury (CI-AKI) is one of the common serious complications of percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS). This study aimed to assess the significance of noncontrast strategy in the setting of ACS. Methods : CI-AKI was defined as an increase in serum creatinine of ≥ 0.5 mg / dL or ≥ 1.25 times from the baseline. One-year worsening renal function (WRF) was defined as an increase of ≥ 0.3 mg / dL in serum creatinine from the baseline after PCI. Results : Of 250 ACS patients, 81 were treated with noncontrast PCI. The average doses of contrast medium in the noncontrast and conventional groups were 17 (9–22) ml and 150 (120–200) ml, respectively. CI-AKI was observed in 4 patients (5%) in the noncontrast group and 29 patients (17%) in the conventional group. Noncontrast PCI was associated with a lower incidence of CI-AKI (adjusted odds ratio, 0.26 ; 95% confidence interval [CI], 0.08–0.82). The bootstrap method and inverse probability weighting led to similar results. CI-AKI was associated with a higher incidence of 1-year WRF (adjusted hazard ratio, 2.30 ; 95% CI, 1.12–4.69), while noncontrast PCI was not. Conclusions : Noncontrast PCI was associated with the lower incidence of CI-AKI in ACS patients

A new protocol for the preparation of superconducting KBi2

A superconducting KBi2 sample was successfully prepared using a liquid ammonia (NH3) technique. The temperature dependence of the magnetic susceptibility (M/H) showed a superconducting transition temperature (Tc) as high as 3.6 K. In addition, the shielding fraction at 2.0 K was evaluated to be 87%, i.e., a bulk superconductor was realized using the above method. The Tc value was the same as that reported for the KBi2 sample prepared using a high-temperature annealing method. An X-ray diffraction pattern measured based on the synchrotron X-ray radiation was analyzed using the Rietveld method, with a lattice constant, a, of 9.5010(1) Å under the space group of Fd[3 with combining macron]m (face-centered cubic, no. 227). The lattice constant and space group found for the KBi2 sample using a liquid NH3 technique were the same as those reported for KBi2 through a high-temperature annealing method. Thus, the superconducting behavior and crystal structure of the KBi2 sample obtained in this study are almost the same as those for the KBi2 sample reported previously. Strictly speaking, the magnetic behavior of the superconductivity was different from that of a KBi2 sample reported previously, i.e., the KBi2 sample prepared using a liquid NH3 technique was a type-II like superconductor, contrary to that prepared using a high-temperature annealing method, the reason for which is fully discussed. These results indicate that the liquid NH3 technique is effective and simple for the preparation of a superconducting KBi2. In addition, the topological nature of the superconductivity for KBi2 was not confirmed

The Molecular Outflows in the rho Ophiuchi Main Cloud: Implications For Turbulence Generation

We present the results of CO (J=3-2) and CO (J=1-0) mapping observations toward the active cluster forming clump, L1688, in the rho Ophiuchi molecular cloud. From the CO (J=3-2) and CO (J=1-0) data cubes, we identify five outflows, whose driving sources are VLA 1623, EL 32, LFAM 26, EL 29, and IRS 44. Among the identified outflows, the most luminous outflow is the one from the prototypical Class 0 source, VLA 1623. We also discover that the EL 32 outflow located in the Oph B2 region has very extended blueshifted and redshifted lobes with wide opening angles. This outflow is most massive and have the largest momentum among the identified outflows in the CO (J=1-0) map. We estimate the total energy injection rate due to the molecular outflows identified by the present and previous studies to be about 0.2 L_solar, larger than or at least comparable to the turbulence dissipation rate [~(0.03 - 0.1) L_solar]. Therefore, we conclude that the protostellar outflows are likely to play a significant role in replenishing the supersonic turbulence in this clump.Comment: 37 pages, 9 figures, accepted for publication in The Astrophysical Journa

EDITORIAL

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting α_vβ₃/α_vβ₅ integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, “cyclic-Arg-Ala-Asp” (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred <i>via</i> an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma

光と酸素或いは過酸化水素を用いる酸化反応の開発に関する研究

酸化反応は有機合成上有用な反応であるが、従来の手法では毒性の高い重金属酸化剤や原子効率の低い複雑な有機分子が必要であった。一方、分子状酸素や過酸化水素を末端酸化剤とする手法は、原子効率が高く、廃棄物として理論上水のみを排出する、グリーンケミストリーの概念に合致した酸化法である。筆者らは最終酸化剤としての「過酸化水素」ならびに「分子状酸素」を利用した環境負荷低減を指向した酸化反応の開発を行った。その結果、ハロゲンソース存在下、スチレン類の光酸素酸化により、対応するフェナシルハライド類を合成することに成功した。また、光酸素酸化反応の後に単体ヨウ素を触媒とする反応をワンポットで行うことにより、スチレン類からアセトフェノン類を、ベンジルアルコール類からビスインドリルメタン類をそれぞれ合成することに成功した。さらに、単体ヨウ素存在下、酸化剤として過酸化水素或いは分子上酸素を用いることにより、三級アミン類と炭素求核剤の酸化的カップリング反応にも成功した。Oxidation is one of the most important reactions in organic synthesis; however, classical methods require toxic heavymetal reagents or complex organic molecules. On the other hand, oxidation using oxygen or hydrogen peroxide has received muchattention in organic synthesis recently since these reagents are effective oxidants of larger atom efficiency and theoretically produceonly water as the end product. With this perspective, we have studied oxidation using oxygen or hydrogen peroxide as a terminaloxidant. As a result, we found that styrenes can be oxidized to corresponding phenacylhalides under aerobic photo-oxidativeconditions in the presence of halogen sources. We also developed one-pot synthesis of acetophenones and bis-indolylmethanes fromstyrenes and benzylalcohols, which includes aerobic photo-oxidation followed by iodine catalyzed reaction, respectively.Furthermore, we developed a cross-dehydrogenative coupling reaction between tertiary amines and carbon nucleophiles usinghydrogen peroxide or oxygen as a terminal oxidant in the presence of catalytic iodine

Evidence For Cloud-Cloud Collision and Parsec-Scale Stellar Feedback Within the L1641-N Region

We present high spatial resolution $^{12}$CO ($J=1-0$) images taken by the Nobeyama 45m telescope toward a $48' \times 48'$ area including the L1641-N cluster. The effective spatial resolution of the maps is $21"$, corresponding to 0.04 pc at a distance of 400 pc. A recent 1.1 mm dust continuum map reveals that the dense gas is concentrated in several thin filaments. We find that a few dust filaments are located at the parts where $^{12}$CO ($J=1-0$) emission drops sharply. Furthermore, the filaments have two-components with different velocities. The velocity difference between the two-components is about 3 km s$^{-1}$, corresponding to a Mach number of 10, significantly larger than the local turbulent velocity in the cloud. These facts imply that the collision of the two components (hereafter, the cloud-cloud collision) possibly contributed to the formation of these filaments. Since the two components appear to overlap toward the filaments on the plane of the sky, the collision may have occurred almost along the line of sight. Star formation in the L1641-N cluster was probably triggered by such a collision. We also find several parsec-scale CO shells whose centers are close to either the L1641-N cluster or V 380 Ori cluster. We propose that these shells were created by multiple winds and/or outflows from cluster YSOs, i.e., "protocluster winds." One exceptional dust filament located at the western cloud edge lies along a shell; it is presumably a part of the expanding shell. Both the cloud-cloud collision and protocluster winds are likely to influence the cloud structure and kinematics in this region.Comment: 44 pages, 12 figures, submitted to Ap