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Tumor biomarkers are highly desirable for the screening of patients with a risk of tumor development and progression. Here, we report a beta-galactosidase (β-gal)-responsive acetaminophen (β-GR-APAP) as a synthetic plasma biomarker for targeted tumor detection. Tumor β-gal labeling via the recognition of tumor-related antigen enabled the detection of a tumor using β-GR-APAP.

Aminopeptidase N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1-(13) C]Ala-NH2 which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN-induced chemical shift change. The [1-(13) C]Ala-NH2 probe allowed sensitive detection of APN activity using (13) C NMR spectroscopy.

Herein, we report on a new (19)F MRI probe for the detection and imaging of H2O2. Our designed 2-fluorophenylboronic acid-based (19)F probe promptly reacted with H2O2 to produce 2-fluorophenol via boronic acid oxidation. The accompanying (19)F chemical-shift change reached 31 ppm under our experimental conditions. Such a large chemical-shift change allowed for the imaging of H2O2 by (19)F chemical-shift-selective MRI.

Hyperpolarization is an emerging method that dramatically enhances NMR signal intensity. As a result of their increased sensitivity, hyperpolarized (HP) NMR molecular probes can be used to perform time-resolved spectroscopy and imaging in vitro and in vivo. It is, however, challenging to design such probes de novo. Herein, the [1

Nuclear hyperpolarization is a phenomenon that can be used to improve the sensitivity of magnetic resonance molecular sensors. However, such sensors typically suffer from short hyperpolarization lifetime. Herein we report that [15N, D14]trimethylphenylammonium (TMPA) has a remarkably long spin–lattice relaxation time (1128 s, 14.1 T, 30 °C, D2O) on its 15N nuclei and achieves a long retention of the hyperpolarized state. [15N, D14]TMPA-based hyperpolarized sensor for carboxylesterase allowed the highly sensitive analysis of enzymatic reaction by 15N NMR for over 40 min in phophate-buffered saline (H2O, pH 7.4, 37 °C).

Adsorption of cellulase onto the saccharification residues of lignocellulose, especially onto lignin, is unproductive and poses a significant problem for recycling of the enzyme. We developed a direct method to quantify residue-adsorbed cellulase by elemental analysis without use of calorimetric methods such as the Bradford, BCA, or Lowry assays. First, initial cellulase loading was estimated from the absorbance at 280 nm using the weight adsorption coefficient of cellobiohydrolase I (CBHI), predicted using the amino acid sequence. Secondly, small amounts of nitrogen derived from adsorbed cellulase were detected using an elemental analyzer by analyzing various large quantities of residue. The nitrogen content of the Trichoderma reesei cellulolytic enzymes was calculated to be 16.5–17.3% based on the amino acid sequences, and the quantity of residue-adsorbed cellulase was calculated from the nitrogen content of the residue, using the nitrogen content of CBHI (16.5%). For example, following enzymatic saccharification of ball-milled softwood for 72 h with Accellerase 1500 (25 filter paper units/g substrate), it was possible to estimate that 18–28 mg cellulase/g of residue was adsorbed onto the residues, indicating that 19–28% of the initially added cellulase would be difficult to recover because of adsorption.

Ca(2+) is a fundamental metal ion for physiological functioning. Therefore, molecular probes for Ca(2+) analysis are required. Recently, a hyperpolarized NMR probe has emerged as a promising tool. Here, we report a new design of a hyperpolarized NMR probe for Ca(2+), which showed a large chemical shift change upon binding to Ca(2+) and was applied for Ca(2+) sensing in a hyperpolarized state.

The cost of cellulase is a major factor limiting the enzymatic hydrolysis of cellulosic biomass. Thus, immobilization of cellulase would be an important advancement. Lignophenol is a lignin-based functional phenolic polymer synthesized from a lignocellulosic material and a phenol derivative at ambient temperature. Cellulase derived from Trichoderma reesei is easily immobilized by softwood and hardwood lignocresol simply by mixing to produce a water-insoluble lignophenol-cellulase complex. Enzymatic hydrolysis performances of cellulases immobilized on lignocresols are approximately 80–90% and 30–50% relative to that of free cellulase in the hydrolysis of carboxymethylcellulose (CMC) and filter paper, respectively. Cellulase was active enough even after adsorption on lignocresols. Limited physical contact between solid substrates and immobilized cellulase due to the presence of lignocresol seems to lead to lower enzymatic activity for solid substrates. Hardwood lignocresol-immobilized cellulase exh...

Steam-exploded eucalyptus wood chips were treated with p-cresol and 72% sulfuric acid at ambient temperature. Steam-exploded lignin was isolated as acetone-soluble and diethyl ether-insoluble compounds from the cresol layer. The lignin extraction yield was only 47%, and the amount of cresol grafted to lignin was much less than that in the case of eucalyptus lignin without steam explosion. Clearly, the steam explosion process depolymerized native lignin, and simultaneously, promoted polymerization via labile benzyl positions. The steam-exploded eucalyptus lignin adsorbed more Trichoderma reesei cellulase; however, its enzymatic activity was less than that of eucalyptus lignin that did not undergo steam explosion. It is evident that pretreatment potentially affects the affinity between lignin and cellulase and the resultant saccharification efficiency.

A model lignin-binding cellulase was prepared from Trichoderma reesei cellulase and lignocresol, which was synthesized from softwood or hardwood lignin. Filter paper was incubated with the lignocresol-cellulase complex, and it was observed that only a limited amount of cellulase migrated to the filter paper. The cellulase adsorption isotherms for the lignocresols and filter paper were fitted to a Langmuir absorption model, and the determined Langmuir constants were as follows: softwood lignocresol>hardwood lignocresol>>filter paper. The calculations demonstrated that lignin-binding cellulase can potentially be recovered by the addition of a sufficient quantity of cellulosic substrate. As a result, the lignocresol-binding cellulase is highly stable and lignocresol can potentially be used for immobilizing cellulase in the active state.

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Monoamine oxidase (MAO) A is a flavoenzyme that catalyzes the oxidation of biologically important monoamines and is thought to be associated with psychiatric disorders. Here, we report a strategy for rationally designing a (19)F magnetic resonance imaging probe for the specific detection of human MAO-A (hMAO-A) activity. Our designed (19)F probe was oxidized expeditiously by hMAO-A to produce 2-fluoro-4-nitrophenol via a spontaneous β-elimination mechanism. Concomitant with the structural change of the probe to the product, the (19)F chemical shift changed by 4.2 ppm, which was enough to visualize the probe and enzymatic product separately. Importantly, our probe achieved excellent discrimination of hMAO-A from its isoform hMAO-B.

A series of water-soluble bis- and tetrakis-ZnII–cyclen complexes with rigid structures was prepared to enhance the carboxylate and phosphate ion binding response in contrast to analogues with less-confined molecular structures. Boc-protected 6-chloro-1,3,5-triazine–bis-cyclen was coupled to different aryl and alkyl moieties in moderate to high yields and subsequently converted into the corresponding bis- or tetrakis-ZnII–cyclen complexes. The bis-ZnII–cyclen moiety is known for its affinity to anions. Depending on the arene substituent some of the synthesized synthetic receptors are luminescent. They were studied by absorption and emission spectroscopy for their response to the presence of phosphate anions of biological relevance in buffered aqueous solution at neutral pH and for their affinity to the genetically encodable oligoaspartate and glutamate sequences (D4 and E4 tag) recently introduced. The rigid structures of the compounds enhance the electronic coupling between the metal complex binding site and the reporter dye. This leads to an increased anion binding response in homogeneous aqueous solution.

Lignophenol is a novel lignin-based functional polymer. Hydrothermal decomposition of ligno- p -cresol derived from softwood (Douglas fir) was studied from 300 to 400 °C as a candidate technology for modification of the molecular structure and the recovery of monophenols. Ligno- p -cresol was promptly depolymerized probably by hydrolysis of aryl ether linkages and the half was converted to ether-soluble compounds at 300 °C for 10 min. With increasing temperature and reaction time, the yields of char and gas were increased, indicating the acceleration of carbonization and decomposition of monophenols. The monophenols obtained were mainly p -cresol and guaiacol. Their yields only exceed 10% at 365 °C and 60 min probably due to the inefficient pyrolysis of 1,1-bis aryl structures.

Enzymatic hydrolysis of cellulosic biomass by cellulase has been receiving attention. Since the cost of cellulase has a big impact on the price of the products, it is important to develop cheaper alternatives. Here we report on the production of a novel immobilized cellulase. A functional lignin-based phenolic polymer “lignophenol” (p-cresol type : ligno-p-cresol) was synthesized from Hinoki wood meal and p-cresol through the phase separation system developed for selective separation of lignocellulosic components. Commercial cellulase derived from Trichoderma reeseiwas easily immobilized on lignophenol simply by mixing to give water-insoluble cellulase-lignophenol complex. Immobilization reached more than 100 mg/g-lignophenol with an enzymatic activity of about 60% compared to free cellulase. With lower immobilization loads, cellulase exhibited lower activity. Cellulase did not significantly detach from lignophenol by mixing with acetate buffer, indicating that the cellulase immobilized by a simple physical adsorption is stable.

Lignophenol is a lignin-based polymer which highly conserves the native lignin structure. Through simple adsorption experiments between Trichoderma reesei cellulase and softwood lignophenol synthesized from Hinoki cypress and p-cresol, several ideas were obtained on non-productive cellulase binding to lignin expected during enzymatic saccharification of cellulosic substrates. Cellulase is promptly bound to lignin within 10 min. The adsorption reaches a saturated value, indicating that cellulase adsorption on lignin is not multi-layered. Maximum adsorption is observed at around the isoelectric point of cellulase and, at the same time, phenolic hydroxyl groups in lignin seem to enhance cellulase binding. Cellulase bound to lignin maintains its enzymatic activity and probably functions as an immobilized cellulase in saccharification liquor.

Mouse lactate dehydrogenase-X (mLDH-X) was designed as a triple-resonance NMR reporter protein. The mLDH-X reporter catalyzes the conversion of a 13C-labeled pyruvic acid derivative (substrate) to ...

Lignocellulosic materials are composed of cellulose, hemicellulose and lignin. Since cellulose is surrounded by lignin, a pretreatment is required to enhance the contact with enzyme for enzymatic hydrolysis. So far, several methods: grinding, steaming, steam explosion, microwave treatment and so on have been studied for wood, however, it is still difficult to saccharify softwood very efficiently. In this study, soda and kraft cooking for chemical pulping were applied for the pretreatment of softwood (Japanese cedar) shaving. The lignin content of kraft pulp ranged from 12% to 3% with increasing cooking time from 1 to 3 hours. Saccharification efficiency after 1 day increased from 90% to 98% with decreasing the lignin content of kraft pulp. The soda and kraft pulp with the same lignin content showed similar saccharification efficiencies. The longer cooking time realized the smaller lignin content, but at the same time, the loss of carbohydrates was larger. As a result, the yields of sugar to wood were almost constant (40%) with a lignin content up to 10% whether the pretreatment was soda or kraft cooking. Even soda cooking is useful enough as a pretreatment method for saccharification of softwood.

Fluorescence sensing with small molecular chemosensors is a versatile technique for elucidation of function of various biological substances. We now report a new fluorescent chemosensor for nucleoside polyphosphates such as ATP using metal-anion coordination chemistry. The chemosensor 1-2Zn(II) is comprised of the two sites of 2,2'-dipicolylamine (Dpa)-Zn(II) as the binding motifs and xanthene as a fluorescent sensing unit for nucleoside polyphosphates. The chemosensor 1-2Zn(II) selectively senses nucleoside polyphosphates with a large fluorescence enhancement (F/F(o) > 15) and strong binding affinity (K(app) approximately = 1 x 10(6) M(-1)), whereas no detectable fluorescence change was induced by monophosphate species and various other anions. The 'turn-on,' fluorescence of 1-2Zn(II) is based on a new mechanism, which involves the binding-induced recovery of the conjugated form of the xanthene ring from its nonfluorescent deconjugated state which was formed by an unprecedented nucleophilic attack of zinc-bound water. The selective and highly sensitive ability of 1-2Zn(II) to detect nucleoside polyphosphates enables its bioanalytical applications in fluorescence visualization of ATP particulate stores in living cells, demonstrating the potential utility of 1-2Zn(II).

Due to the difficulty of multiple deletions using the Cre/ loxP system, a simple, markerless multiple-deletion method based on a Cre / mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum . Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/ loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum . It should facilitate the understanding of genome functions of microorganisms.

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.

Hydrothermal treatment can be applied to pretreatment of ethanol fermentation process. What is important in the pretreatment step is that lignocellulosic material be effectively hydrolyzed to smaller saccharides and not be further converted to other substances. In this study we developed reaction kinetics of cellulose hydrolysis to glucose for evaluation of hydrothermal treatment condition.

症例は69歳, 女性. 主訴は残尿感, 下腹部違和感. 慢性腎不全のため平成5年から維持血液透析を受けていた. 平成14年5月下旬頃から残尿感・下腹部違和感などの膀胱炎症状を1-2か月に1度の頻度で繰り返していた. 尿路感染では違和感・残尿感が続くが, 無尿のため排尿できず, 非常に強い苦痛を伴っていた. 尿路感染に対し抗菌薬を投与するも効果が不十分なため, 膀胱洗浄を併用しなければならない場合もあった. 繰り返す難治性尿路感染症の治療目的で清心蓮子飲を投与したところ, 抗菌薬の効果が増強し, 最終的には抗菌薬を投与しなくても尿路感染が再発しなくなった. 無尿の患者に清心蓮子飲が有効であったことから, 清心蓮子飲が血行性に抗菌効果を増強させている可能性が示唆された. 本論では清心蓮子飲の作用機序について, 古典の記述と構成生薬の2点から検討した.

高温高圧環境下での電気化学測定を可能とした測定セルを用いて，メタノール濃度0.001–1mol kg−1，温度343–523Kの高温水中で，メタノール電極酸化反応の定常的な分極特性を測定し，反応の活性化エネルギーやメタノールについての総括の反応次数を求めた．0.4V vs. RHE以下の低電位において，活性化エネルギーは，メタノール濃度0.1mol kg−1のとき60kJmol−1であったのに対し，1mol kg−1のときは30kJmol−1台の小さい値をとった．一方，0.5V vs. RHE以上の電位では，本実験の測定濃度において，活性化エネルギーは60kJmol−1で一致し，メタノールについての総括の反応次数は0.4と算出された．

A Zn(II) complex (Zn(II)-Ida) was designed as the new fluorescent probe for His-tag fused proteins. Thanks to the tight binding ability to histidine-rich sequences and bright fluorescence property of the Cy5-appended Zn(II)-Ida probes, selective and clear fluorescent imaging of the His-tag fused G-protein coupled receptors on live cell surfaces was carried out.

The first dual activatable hypochlorite ((-)OCl)-sensing probe was developed, based on a new proof-of-concept design involving signal-activatable (1)H chemical probes using the triple-resonance NMR technique. The probe enabled fluorescence-(1)H MR dual turn-on detection of (-)OCl in solution and in crude tissue extracts.

Electrochemical oxidation of carbon monoxide between 0.3 and 0.5 V vs. RHE (reversible hydrogen electrode) was investigated in hot 0.005 M H2SO4–0.2 M Na2SO4 solution at 423 and 473 K under CO pressures ranging from 0.08 to 1.25 MPa. The reaction could be well explained by both the Langmuir–Hinshelwood mechanism for the reaction between carbon monoxide and water and the Langmuir isotherm for adsorption of carbon monoxide on a Pt electrode. Activation energy around 60 kJ·mol–1 was observed. This value almost agrees with the apparent activation energy for electrochemical oxidation of methanol on a Pt electrode obtained between 393 and 463 K by Watanabe et al. (1997). We confirmed that CO adsorption decreases with the increase of temperature.

In order to survey the current status of home care and support for patients with hematological diseases, questionnaires were sent to 3,591 hospitals and home care facilities in Tokyo and surrounding prefectures. The first survey showed that 81.7% of medical staff members at hospitals reported that they had experience with home care and support, but only 24.9% of home care facility staff members had such experience. The second questionnaire, surveying 1,202 personnel, identified four factors hampering successful establishment of home care and support networks for hematological diseases. These included insufficient familial support for patients, difficulty making end of life decisions by family members and patients, limited access to transfusion support, and financial problems.

The carbide skiving hob was developed by Dr. Masato Ainoura in 1963 with the objective of finishing only the tooth surface of hardened gears. It is currently use in many countries in worldwide including Japan. In carbide skiving, only the side cutting edge works, and the top of its tooth doesn't. Thus when a tooth surface of the Shore hardness 85HS, is finished or when a cutting edge can hardly cut because of the large wear on it, a hob is apt to shift to the axial direction and a gear is forced to rotate. Here we would like to report a countermeasure (the modified pithed hob for finishing hardened gears) for the imbalance of cutting resistance on cutting edges at the right and left sides.

We report a new strategy for designing a signal off-to-on-type (19)F MRI chemical probe that operates in biological environments. The present strategy is based on the control of adherence of a (19)F MRI chemical probe to certain blood proteins, accompanied by a change in transverse relaxation time of (19)F nuclei.

A functional lignin-based material, lignocresol, was synthesized from eucalyptus wood meal by using phase separation treatment with p-cresol and 72% sulfuric acid. Lignocresol physically adsorbs Trichoderma reesei cellulase and functions as an immobilized cellulase. Lignocresol-immobilized cellulase was treated with acetone or ethanol to separate the enzyme and its support. Most lignocresol was dissolved in organic solvents and then precipitated in diethyl ether. The recovery yield of lignocresol using acetone was 85% and that using ethanol was 72%, with very low or negligible residual cellulase. Lignocresol is an innovative recyclable enzyme support that can be easily separated using only organic solvents after deactivation of the immobilized enzyme.