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The alternative oxidase (AOX) is a monotopic diiron carboxylate protein which catalyzes the four-electron reduction of dioxygen to water by ubiquinol. Although we have recently determined the crystal structure of Trypanosoma brucei AOX (TAO) in the presence and absence of ascofuranone (AF) derivatives (which are potent mixed type inhibitors) the mechanism by which ubiquinol and dioxygen binds to TAO remain inconclusive. In this article, ferulenol was identified as the first competitive inhibitor of AOX which has been used to probe the binding of ubiquinol. Surface plasmon resonance reveals that AF is a quasi-irreversible inhibitor of TAO whilst ferulenol binding is completely reversible. The structure of the TAO-ferulenol complex, determined at 2.7 A, provided insights into ubiquinol binding and has also identified a potential dioxygen molecule bound in a side-on conformation to the diiron center for the first time.

Pharmacological inhibition of histone deacetylase 6 (HDAC6) is an effective therapeutic strategy for cancer and immunological diseases. Most of the previously reported HDAC6 inhibitors have a hydroxamate group as a zinc binding group (ZBG), which coordinates to the catalytic zinc ion of HDAC6. The hydroxamate group is liable to metabolically generate mutagenetic hydroxylamine; therefore, non-hydroxamate HDAC6 inhibitors would be advantageous. In this study, to identify novel non-hydroxamate HDAC6-selective inhibitors, screening of a chemical library and the subsequent structural optimization were performed, which led to the identification of HDAC6-selective inhibitors with 3,3,3-trifluorolactic amide (TFLAM) as a novel ZBG. The identified inhibitor showed potent and selective HDAC6-inhibitory activity in cells and induced regulatory T (Treg) cell differentiation.

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been proven to have clinical importance and has been manufactured industrially since 1993 by using mutants with high FK506-production ability; these mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production. However, there was no effective industrial method to separate FR900525 from FK506 due to the structural similarity between the two compounds. Therefore, reducing the level of FR900525 was a serious problem in the industrial strain A. In this study, we aimed to reduce the FR900525 production. We first determined that pipecolic acid level was a critical parameter for controlling FR900525 production in strain A. S-(2-Aminoethyl) l-cysteine (AEC)-resistant mutants has been reported to increase lysine productivity successfully in a variety of lysine-producing microorganisms. Therefore, next, we applied a selection of AEC-resistant mutants to enhance pipecolic acid biosynthesis. Finally, four AEC-resistant mutants were obtained from strain A using ultraviolet irradiation, and three of them showed less FR900525 productivity compared to the parental strain A. Our findings indicated that AEC resistance was effective phenotype marker for increasing pipecolic acid productivity and for reducing FR900525 production in S. tsukubaensis. Thus, our study provides an efficient method for reducing FR90025 level during FK506 biosynthesis.

What N would you like? The chemoselective and complementary Pd- and Cu-catalyzed N-arylation of 2-aminobenzimidazoles is described. Selective N-arylation of the amino group was achieved with a Pd-catalyzed method, while selective N-arylation of azole nitrogen was achieved with a Cu-catalyzed procedure (see scheme)., National Institutes of Health (U.S.) (GM58160)

A new biarylphosphine ligand, Me(3)(OMe)tBuXPhos (L3), was designed as a surrogate for Me(4)tBuXPhos (L1). The Me(3)(OMe)tBuXPhos could be prepared in a chromatography-free manner from inexpensive and readily available 2,3,6-trimethylphenol. Comparative studies demonstrated that a catalyst based on Me(3)(OMe)tBuXPhos displayed the same reactivity as a catalyst based on Me(4)tBuXPhos for Pd-catalyzed C-N and C-O bond-forming processes.

Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.

Highly N2-selective arylation of 4,5-unsubstituted and 4-substituted 1,2,3-triazoles was achieved for the first time by Pd/L1 catalyst system. A wide range of N2-aryl-1,2,3-triazoles were prepared from aryl bromides, chlorides and triflates with excellent (95–99%) N2-selectivity. DFT calculations suggest that formation of N2-arylated 1,2,3-triazoles is favored kinetically.

In two patients with steroid-resistant nephrotic syndrome (SRNS), we investigated the relationship between clinical findings during immunosuppressive therapy and multiple drug resistant gene-1 (MDR-1) expression. MDR-1 was detected by real-time polymerase chain reaction (PCR). In a boy who initially developed SRNS at 3 years, we observed MDR-1 expression over 3 years. Maximal and minimal MDR-1 expression were 90 000 and 7800 copies/µg RNA, respectively. In a 4-year-old boy who initially developed SRNS at 3 years, we determined MDR-1 expression over 2 years. Maximal and minimal MDR-1 expression were 42 000 and 6900, respectively. MDR-1 evaluation requires determination of MDR-1 expression at several time points in a clinical course. Establishment of a normal expression may be needed for each individual patient. Increasing MDR-1 during remission was followed soon by recurrences, an observation that may be a guide for therapeutic choice. LDL influences a humoral factor involved in MDR-1 expression. Both patients responded to LDL adsorption therapy because of elevated LDL levels. While cyclosporine A therapy gradually decreased MDR-1 expression, LDL adsorption therapy decreased expression sharply. Based on the results of the present study, LDL adsorption therapy could contribute to the amelioration of drug sensitivity for immunosuppressants including corticosteroids via inhibitory effects on MDR-1 expression.

The filamentous fungus Coleophoma empetri F-11899 produces an echinocandin-like compound FR901379, the original source for micafungin which is prescribed to treat deep-seated mycoses. Despite its industrial importance, no genetic information on C. empetri F-11899 is currently available. To characterize FR901379 biosynthetic genes by insertional mutagenesis and to improve the compound production genetically, Agrobacterium tumefaciens-mediated transformation (ATMT) was attempted to make genetic manipulation possible in this strain. The optimum conditions for ATMT of C. empetri were determined for the cell density of bacteria, time period of co-cultivation and types of filters in co-cultivation. Using the established ATMT method, the hygromycin B resistant gene was successfully transferred into the genome of C. empetri F-11899 and stably maintained even after a serial passage. Some of these results will be applicable for ATMT of various filamentous fungi.

A new quantitative and chemical-state analysis method to measure oxygen on the surface of graphitic carbon materials by total-electron-yield soft X-ray absorption spectroscopy (TEY-XAS) is proposed, and applied to the analysis of graphite oxides (GO). In this method, working curves for quantitative analysis were successfully obtained from the relative absorption- peak-intensity in the OK and CK absorption edges as functions of the O/C atomic ratio in standard organic samples which have various oxygenated functional groups. The atomic ratio O/C on the GO surface was determined as approximately 0.35 from the working curves, and the chemical states of surface oxygen were estimated as mainly –OH and partially >C=O and/or –CH=O from the finger print method of the TEY-XAS.

Soluble amino acids in roots and primary amino acids, which were involved in primary ammonium assimilation, in the metabolites of 14C-glucose fed to roots for 3 h in the dark were analyzed in the roots of non-nodulated soybean and pea plants grown in ammonium, nitrate or nitrogen-free media for 1 day. Compared with the effect of nitrate, ammonium supply strongly affected the content and synthesis of the amino acids in the roots. In both soybean and pea roots, the supply of ammonium increased considerably the concentrations of the primary amino acids, and asparagine was the most predominant amide, followed by glutamine. In nitrate-supplied soybean roots, the concentrations of asparagine, aspartate and alanine increased, but the concentration of glutamine was low. In the roots of pea plants grown in nitrate media, asparagine was the predominant amino acid, although the composition of the primary amino acids was little affected by nitrate supply. The proportion of amino acids synthesized from 14C-gl...

The chemokine receptor CXCR4 possesses multiple critical functions in normal and pathologic physiology. CXCR4 is a G-protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1). The interaction between CXCL12 and CXCR4 plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), and pulmonary fibrosis. It is conjectured that this interaction may be a critical therapeutic target in all of these diseases, and several CXCR4 antagonists have been proposed as potential drugs. Fourteen-mer peptides, T140 and its analogues, were previously developed in our laboratory as specific CXCR4 antagonists that were identified as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents, and anti-RA agents. Cyclic pentapeptides, such as FC131 [cyclo(D-Tyr-Arg-Arg-L-3-(2-naphthyl)alanine-Gly)], were also previously found as CXCR4 antagonist leads based on pharmacophores of T140. This review article describes the elucidation of multiple functions of CXCR4 antagonists and the development of a number of low-molecular weight CXCR4 antagonists involving FC131 analogues and other compounds with different scaffolds including linear-type structures.

Ni(acac) 2 catalyzes the four-component connection reaction of diphenylzinc, isoprene, aromatic aldehydes, and aromatic amines in this order and provides stereochemically homogeneous ( E )-1-arylamino-1-aryl-3-methyl-5-phenyl-3-pentenes ( 1 ) in excellent yields. Aliphatic aldehydes react similarly and give ( E )-1-arylamino-1-alkyl-3-methyl-5-phenyl-3-pentenes ( 1 ) in slightly reduced yields. When the alkyl groups are bulky, in addition to 1 are formed ( E )-1-arylamino-1-alkyl- 4 - methyl -5-phenyl-3-pentenes ( 1′ ) as the minor products. Lactamines prepared in situ from five- and six-membered lactols and aromatic amines are more reactive than alkyl aldehyde aldimines and furnish ( E )-4-arylamino-6-methyl-8-phenyl-6-octen-1-ols ( 4 ) and ( E )-5-arylamino-7-methyl-9- phenyl-7-nonen-1-ols ( 5 ), respectively, in good yields with excellent E -stereoselectivity.

This paper presents a description of the Hybrid Core Calculation System which is based on a very rigorous but practical method utilizing best estimate core design calculations and taking advantage of the recent remarkable progress of computers. The basic idea of this system is to generate the correction factors for assembly-homogenized cross sections, discontinuity factors, etc. by comparing the CASMO-4 and SIMULATE-3 2-D full-core calculation results under the consistent calculation condition and applying them to the SIMULATE-3 3-D calculation. The CASMO-4 2-D heterogeneous core calculation is performed for each depletion step using the core conditions previously determined by ordinary SIMULATE-3 core calculations. This avoids time-consuming iterative calculations of the critical boron concentration search and the thermal hydraulic feedback. These calculations are instead performed in the final SIMULATE-3 3-D calculation using the previously determined correction factors. The Hybrid Core Calculation Syst...

The goal of this study is to develop a novel sustained-release (SR) system for poorly water-soluble drugs by applying solid dispersion (SD) technique for improving the solubility. The developed SR system, disintegration-controlled matrix tablet (DCMT), consists of hydrogenated soybean oil (HSO) as wax and SD granules containing low-substituted hydroxypropylcellulose (L-HPC) as a disintegrant. In this study, nilvadipine (NiD) was chosen as a model compound. Sustained-release profiles of NiD from DCMT were identically controlled in several dissolution mediums in spite of varying pH and agitation speed. The release of NiD from DCMT was sustained more effectively by increasing the amount of wax or by decreasing the amount of disintegrant, and supersaturation of NiD was achieved without any re-crystallization in dissolution medium. The release rate of NiD from DCMT was controlled by the disintegration rate of tablet. The release profile of NiD was described by the Hixson-Crowell's model better than zero-order kinetics, first-order kinetics and Higuchi's model, which supports that the release of NiD from DCMT is regulated by the disintegration of the tablet. From this study, it was clarified that DCMT was one of the promising SR systems applying SD for the poorly water-soluble drugs.

L,L-Type and L,D-type (E)-alkene dipeptide isosteres (EADIs) that have unnatural side chains at the alpha-position were synthesized by the combination of stereoselective aziridinyl ring-opening reactions and organozinc-copper-mediated anti-S(N)2' reactions toward a single substrate of gamma,delta-cis-gamma,delta-epimino (E)-alpha,beta-enoate. The utility of this methodology was demonstrated by the stereoselective synthesis of a set of diastereomeric EADIs of L-Arg-L/D-3-(2-naphthyl)alanine (Nal) that is contained in a small CXCR4 antagonist FC131 [cyclo(-D-Tyr-Arg-Arg-Nal-Gly-)]. Furthermore, a (Nal-Gly)-type EADI was synthesized by samarium diiodide (SmI(2))-induced reduction of a gamma-acetoxy-alpha,beta-enoate. Several FC131 analogues, in which these EADIs were inserted for reduction of their peptide character, were synthesized with analogues containing reduced amide-type dipeptide isosteres to investigate the importance of these amide bonds for anti-HIV and CXCR4-antagonistic activity.

In order to improve the efficiency in gas chromatographic isotope separation technology, porous SiC supporting palladium -platinum (Pd-Pt) alloy as a column material was developed. It was confirmed that the utilization of the column material developed enabled higher recovery yield of 90 % and the higher purity of 97 % for deuterium isotopic separation comparing with the method using conventional column materials such as the mixture of Pd-Pt alloy filler under 100 mesh and Cu powder from 40 to 80 mesh.

With regard to tritiated contaminants, effective and simple dry decontamination method using ozone gas were proposed. By the treatment in ozone gas of 400 ppmv at 400 K, in the cases of stainless steel, the decontamination efficiencies of over 99 % were obtained for 1 hour, and the decontamination efficiencies of aluminum were 80–86 %. By the treatment of ozone gas, the quantity of the carbon atom on the surface and in inside layer were decreased, and the oxidation on the surface and in inside was proceeded, which may be assumed and expected that the recombination, release and diffusion to the surface of tritium are prevented by a rigid oxide layer generated.The ozone gas treatment is easy to use and apply in practice with a simple and safe operation. Furthermore, gas-phase decontamination technology has many advantages over conventional wet methods, in particular, its simple control processes and small secondary waste. The proposed decontamination technology has the sufficient ability and potenti...

We report here the experimental results suggesting the occurrence of multibody nuclear reactions in metal deuterides under ion-beam irradiation and electrolysis. A meaningful increase of helium-4 was observed during electrolysis with the Pd–D2O system, while neutron emission was not observed. The D+D+D fusion, 3D→t+3He+9.5 MeV, has been observed repeatedly in deuteron-beam irradiation experiments with a TiDx target. On the other hand, in proton-beam experiments with TiDx, H+D+D-fusion: H+D+D→p+4He+23.8 MeV was observed. Considering this result, it seems that the 3D reaction occurred between two deuterons trapped closely in TiDx and an incident particle of deuteron. The multibody nuclear reaction model can interpret both the results obtained in electrolysis and ion-beam experiments. It is considered that the lattice dynamics of metal deuteride is of key importance for inducing short-transient and closely packed d–d pairs and, thus, such fusions.

The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9-1-1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9-1-1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.

A newly developed technique, β-ray induced X-ray spectrometry (BIXS), was applied to in situ and real-time measurements of depth profiles of tritium in palladium and zirconium. Two distinct spectra were detected by BIXS, one due to the characteristic X-rays giving rise to a sharp intense peak; and the other due to the bremsstrahlung X-rays leading to a broad weak peak. The intensities of these peaks for palladium decreased with time at room temperature, while they were almost constant for the Zr-sample at room temperature. Heating the Zr-sample, however, caused similar changes in the spectra as those for palladium at room temperature. It was found that these phenomena are due to the diffusion of tritium into the bulk regions, causing a reduction of the tritium concentration in the sub-surface layers of the samples. All of the observed β-ray induced X-ray spectra could be reproduced well by computer simulation by assuming respective depth profiles of tritium in the samples. It was confirmed that BIXS is quite useful to analyze the dynamic transport behavior of tritium in materials.

In order to examine the relationship between long-term potentiation (LTP) and phosphoinositide (PI) turnover, we evaluated these throughout anesthetized rat brain using carbon-11-labeled diacylglycerol (11C-DAG). High-frequency tetanic stimulation (400 pulses at 400 Hz) to the perforant pathway induced LTP in rat dentate gyrus. In autoradiograms of rat brains, LTP was associated with the occurrence of multiple highly radioactive spots in many regions distant from the stimulated site. Following i.v. administration of an NMDA receptor antagonist prior to stimulation, however, no high-density spots were found. These findings directly demonstrate that potentiation of phosphoinositide-derived signaling was induced during LTP, and the finding of multiple location suggests the occurrence of polysynaptic neurotransmission through neural networks pertaining to learning and memory.

In vivo oral absorption study of time-controlled explosion system (TES), using gastrointestinal (GI) physiology regulated dogs, was carried out to predict the feasibility in humans. TES is characterized by rapid drug release with a pre-programmed lag time, which can provide a programmed release system synchronized with circadian rhythm (e.g. asthma attack in the morning), a colon targeting system and a sustained release system with different lag times. In this study, TES containing diclofenac sodium with different lag times of 3 and 6 h (TES-3h and TES-6h) were prepared. TES-3h exhibited good performance in all six GI physiology regulated dogs without remarkable reduction of AUC. In the case of TES-6h, drug absorption was observed ∼6 h after administration in four of six dogs, but plasma level was low. Further, the location of the dosage forms after oral administration was estimated from the gastric emptying time (GET) and the small intestinal transit time (SITT) using a double marker method. As a result, in vivo performance of TES correlated with the intestinal location. It was concluded that TES-3h would perform well in humans and that the environmental water content in the GI tract affected the in vivo dissolution profile of TES when the drug release was initiated after entering the colon.

Our objective was to assess proton chemical shift imaging for potential clinical application in monitoring response to gamma knife radiosurgery. Twenty-five proton chemical shift imaging studies and conventional magnetic resonance images were performed on six patients with intracranial tumors. The peak areas of N-acetylaspartate, choline-containing compounds (Cho), creatine, and lipids were calculated and normalized to N-acetylaspartate in the contralateral hemisphere. The spectra from the lesion before treatment showed a relatively high Cho peak, reported as a characteristic spectrum of tumors. Tumor size and Cho level after radiosurgery did not increase except in two cases. In these cases, radiation necrosis was observed with elevated Cho and a mobile lipid peak. Stable or decreased Cho seems to suggest a loss of tumor viability, and changes in Cho indicate the effectiveness of radiosurgery. Increasing Cho and the appearance of the mobile lipid peak may distinguish radiation necrosis from recurrent tumors, which cannot be distinguished by magnetic resonance imaging.

Epidermal growth factor receptor (EGFR) plays an important role in the progression of malignancy in gliomas. We studied the growth inhibition of the malignant glioma cell lines using an antisense EGFR oligodeoxynucleotide enveloped with LipofectinR. At a concentration of 5 μM of the antisense EGFR oligodeoxynucleotide enveloped with LipofectinR, the proliferation of three malignant glioma cell lines was significantly inhibited (p < 0.05) compared with that of the cells exposed to 5 μM sense EGFR oligodeoxynucleotide. The activity of the tyrosine kinase and the DNA synthesis was also significantly suppressed (p < 0.05). These findings show that the antisense EGFR oligodeoxynucleotide enveloped with LipofectinR has a possibility to become a useful gene therapy against malignant gliomas.

Seventeen brain tumors were measured by 1H-CSI (chemical shift imaging) in a 1.5 T clinical magnetic resonance scanner. The metabolic peaks obtained were evaluated by two methods. One method was to obtain the percentage of each metabolite relative to the combined choline, creatine and NAA peak areas, and the other method was to obtain a ratio of the tumor to contralateral brain. The percentage of choline (%Cho) and choline ratio increased, and the %NAA and NAA ratio decreased in the gliomas and malignant tumors. In relation to grading, %Cho increased but the choline ratio did not. We believed the reason for this was that there were many foci of microscopic necrosis in the glioma grade IV. Free lipids were observed in most of the high grade gliomas and in a malignant tumor. Lactate increased in higher grade tumors. Meningiomas showed the highest %Cho. Statistical differences between the grades of glioma were not detected because many tumors had heterogeneous tissue. One resolution to this problem was metabolite mapping. Mapping of the percentage of metabolites was suitable because it described the regional metabolic changes and the resulting signal to noise ratio was better than that achieved by other methods of evaluation.

An efficient and simple method for the synthesis of various [1, 2, 3]triazolo[1, 5-a]pyridines has been established. The method involves a copper(II)-catalyzed oxidative N-N bond formation that uses atmospheric oxygen as the terminal oxidant following hydrazonation in one pot. The use of ethyl acetate as the solvent dramatically promotes the oxidative N-N bond-formation reaction and enables the application of oxidative cyclization in the efficient one-pot reaction. A mechanism for the reaction was proposed on the basis of the results of a spectroscopic study.

The chemoselective and complementary Pd-and Cu-catalyzed N-arylation of 2-aminobenzimidazoles is described. Selective N-arylation of the amino-group was achieved with a Pd-catalyzed method, while selective N-arylation of azole nitrogen was achieved with a Cu-catalyzed procedure. The utility of these complementary sets of conditions is demonstrated in several two-step, selective syntheses of di-arylated aminoazoles.

Phospholipid metabolism is closely related to membrane perturbation in cerebral ischaemia. We investigated in vivo topographical lipid metabolism using an iodine-123-labelled diacylglycerol analogue, (1-(15-(4-iodine-123-iodophenyl)-pentadecanoyl)-2-stearoyl-rac-gly cerol) (123I-labelled DAG), in a middle cerebral artery (MCA) occlusion model with the aim of positive imaging of ischaemic insult. Sprague-Dawley rats underwent coagulation of the MCA to induce permanent occlusion. MCA occlusion times prior to injection of 123I-labelled DAG ranged from 15 min to 14 days. Each rat was injected with 11-37 MBq of 123I-labelled DAG via a tail vein. After 30 min, in vivo autoradiographs were reconstructed. Scanning of the living rat brain in this MCA occlusion model was performed using a gamma camera with a pinhole collimator. Cerebral infarctions were recognized in the frontal cortex, the parietal cortex and the lateral portion of the caudate-putamen by 2,3,5-triphenyltetrazolium hydrochloride staining. In infarcted regions (region 1), 123I-labelled DAG incorporation showed a slight decrease up to 12 h; it then increased up to 6 days and decreased thereafter. In peri-infarcted regions (region 2), the incorporation showed almost no change up to 12 h, then increased up to 5-6 days and decreased thereafter. In other regions (region 3), the incorporation showed no change. Lipid analysis showed that 123I-labelled DAG was metabolized to 15-(4-iodine-123-iodophenyl)-pentadecanoic acid by DAG lipase and to 123I-labelled phosphatidylcholine. Scanning of the ischaemic region showed higher accumulation than on the non-lesioned side. We established a method to visualize ischaemic foci as positive images. The early changes in 123I-labelled DAG incorporation were closely related to DAG lipase, which degraded the accumulated intrinsic DAG, and increased 123I-labelled DAG incorporation in the chronic stage involves several aspects of neural destruction in the process of autolysis. It is concluded that the reported method could have a clinical future.

The new and inexpensive phosphine promotes the Pd-catalyzed arylation of nitrogen and oxygen nucleophiles and shows the same effectivity as Me4tBuXPhos.

The completely N1-selective Pd-catalyzed arylation of unsymmetric imidazoles with aryl halides and triflates is described. This study showed that imidazoles have a strong inhibitory effect on the in situ formation of the catalytically active Pd(0)–ligand complex. The efficacy of the N-arylation reaction was improved drastically by the use of a preactivated solution of Pd2(dba)3 and L1. From these findings, it is clear that while imidazoles can prevent binding of L1 to Pd, once the ligand is bound to the metal, these heterocycles do not displace it. The utility of the present catalytic system was demonstrated by the regioselective synthesis of the clinically important tyrosine kinase inhibitor nilotinib.

Arylation of 1,2,3-triazoles with aryl halides is achieved with excellent N2-selectivity in the in the presence of a novel Pd—biphenylphosphine catalyst.

To describe lag time of Time-Controlled Explosion System (TES), the system's water absorption kinetics was investigated. Study of water absorption in TES with EC membrane revealed the following relations : (i) the square of water-uptake linearly increased with time in accordance with the Washburn equation, (ii) the water permeation rate correlated with the reciprocal of membrane thickness, (iii) the amount of water-uptake necessary for membrane destruction was directly proportional to the thickness of the membrane. These results suggest that lag time of TES is regulated by the function of membrane thickness. Indeed, the observed lag time was confirmed to fit with the curve expected by the above relations. When talc was included in the EC membrane in an equal weight ratio as a membrane-filler, lag time related directly to the membrane thickness. Compared with TES consisting of EC membrane only, the membrane was destroyed by a smaller amount of uptaken water owing to the weakening of membrane strength by the talc addition.

脳虚血に対する塩酸ビフェメラン (Bifemerane hydrochloride) の効果を実験的に検討した.Pulsinelli等の報告によるラット主幹4動脈閉塞による前脳虚血モデルを用いた.30分間の4動脈の閉塞の後, 両側の総頸動脈の血流を再開し, その後の急性期の脳内エネルギー代謝の状態を31P-magnetic resonance spectrum (MRS) を測定する事により, in vivoで, かつ非侵襲的に観察した.さらに, その後のラットの生存期間を測定した.塩酸ビフェメラン投与群と非投与群とで結果を比較検討した.平均生存期間は非治療群では4.8日であったが, 塩酸ビフェメラン投与群では, 12.8日に延長した.31P-MRS測定の結果, 血流再開により早期からエネルギー代謝は急速な回復を示したが, 塩酸ビフェメランにより, このエネルギー代謝の急激な回復という変化が抑制される傾向が認められた.脳内エネルギー代謝の回復過程への効果と, 生存期間の延長という結果との間の関連に関しての詳細は不明な点が多いが, 塩酸ビフェメランの治療効果を客観的に評価する結果として興味あるものと考えられる.

A novel controlled drug release system. Time-Controlled Explosion System (TES) has been developed. TES has a four-layered spherical structure, which consists of core, drug, swelling agent and water insoluble polymer membrane. TES is characterized by a rapid drug release with a precisely programmed lag time; i.e. expansion of the swelling agent by water penetrating through the outer membrane, destruction of the membrane by stress due to swelling force and subsequent rapid drug release. For establishing the concept and development strategy, TES was designed using metoprolol and polystyrene balls (size: 3.2 mm in diameter) as a model drug and core particles. Among the polymers screened, low-substituted hydroxypropylcellulose (L-HPC) and ethylcellulose (EC) were selected for a swelling agent and an outer water insoluble membrane, respectively. The release profiles of metoprolol from the system were not affected by the pH of the dissolution media. Lag time was controlled by the thickness of the outer EC membrane; thus, a combination of TES particles possessing different lag times could offer any desired release profile of the model compound, metoprolol.

The completely N(1)-selective Pd-catalyzed arylation of unsymmetric imidazoles with aryl halides and triflates is described. This study showed that imidazoles have a strong inhibitory effect on the in situ formation of the catalytically active Pd(0)-ligand complex. The efficacy of the N-arylation reaction was improved drastically by the use of a preactivated solution of Pd(2)(dba)(3) and L1. From these findings, it is clear that while imidazoles can prevent binding of L1 to Pd, once the ligand is bound to the metal, these heterocycles do not displace it. The utility of the present catalytic system was demonstrated by the regioselective synthesis of the clinically important tyrosine kinase inhibitor nilotinib.

The back bombardment (BB) effect limits wide usage of thermionic rf guns. The BB effect induces not only ramping-up of a cathode's temperature and beam current, but also degradation of cavity voltage and beam energy during a macropulse. This paper presents a comparison of the BB effect for the case of dispenser tungsten-base (DC) and lanthanum hexaboride (${\mathrm{LaB}}_{6}$) thermionic rf gun cathodes. For each, particle simulation codes are used to simulate the BB effect and electron beam dynamics in a thermionic rf gun cathode. A semiempirical equation is also used to investigate the stopping range and deposited heat power of BB electrons in the cathode material. A numerical simulation method is used to calculate the change of the cathode temperature and current density during a single macropulse. This is done by solving two differential equations for the rf gun cavity equivalent circuit and one-dimensional thermal diffusion equation. High electron emission and small beam size are required for generation of a high-brightness electron beam, and so in this work the emission properties of the cathode are taken into account. Simulations of the BB effect show that, for a pulse of $6\text{ }\text{ }\ensuremath{\mu}\mathrm{s}$ duration, the DC cathode experiences a large change in the temperature compared with ${\mathrm{LaB}}_{6}$, and a change in current density 6 times higher. Validation of the simulation results is performed using experimental data for beam current beyond the gun exit. The experimental data is well reproduced using the simulation method.

Natural fermentation products have long been studied as attractive targets for drug discovery due to their amazing diverse, complex chemical structures and biological activities. As such, a number of revolutionary drugs developed from natural fermentation products have contributed to global human health. To commercialize a drug derived from natural fermentation products, an effective chemical entity must be identified and thoroughly researched, and an effective manufacturing process to prepare a commercial supply must be developed. To construct such a manufacturing process for tacrolimus and micafungin, the following studies were conducted: first, we focused on controlling the production of the tacrolimus-related compound FR900525, a fermentation by-product of tacrolimus which was critical for quality assurance of the drug substance. FR900525 production was reduced by using a mutant strain which produced more pipecolic acid, the biosynthesis material of tacrolimus, than the original strain. Then, to optimize the fermentation process of FR901379, an intermediate of micafungin, a fed-batch culture was adopted to increase FR901379 productivity. Additionally, FULLZONE(TM) impeller was installed into the scaled-up fermenter, reducing the agitation-induced damage to the mycelium. As a result, the mycelial form changed from filamentous to pellet-shaped, and the air uptake rate during fermentation was drastically improved. Finally, we conducted screening for FR901379 acylase-producing microorganisms, as FR901379 acylase is necessary to manufacture micafungin. We were able to easily discover FR901379 acylase-producing microorganisms in soil samples using our novel, convenient screening method, which involves comparing the difference in antibiotic activity between FR901379 and its deacylated product.

Brain edema can be classified into three categories: vasogenic, cytotoxic, and interstitial. The mechanism of edema is thought to be different in each type. The authors studied the movement of water molecules in each type of white matter edema in a rat model by using diffusion-weighted magnetic resonance imaging. Conventional T2-weighted imaging did not allow distinction between the three types of white matter edema; the three types of edema were, however, distinguished by using diffusion-weighted imaging. The apparent diffusion coefficient (ADC) of water was different in each type of edema. Water molecules in cytotoxic edema induced by triethyl-tin intoxication showed a smaller and less anisotropic ADC than in normal white matter. In contrast, water in vasogenic edema induced by cold injury had a larger and more anisotropic ADC than in normal white matter. Water in interstitial edema due to kaolin-induced hydrocephalus had an anisotropic and very large ADC.

FR901379 acylase, an enzyme that catalyzes the hydrolysis of the palmitoyl moiety of the antifungal lipopeptide FR901379, was purified from the culture broth of Streptomyces sp. no. 6907 (FERM BP-5809), revealing the 80 kDa, two-subunit heterodimeric protein characteristic of the β-lactam acylase family. Using oligodeoxyribonucleotide primers constructed on the basis of the N-terminal amino acid sequence of each purified subunit, the gene was identified from a cosmid library of Streptomyces sp. no. 6907 DNA. The deduced 775 amino acid sequence corresponded to a single polypeptide chain containing two subunits, and it shared 41.7% identity with aculeacin A acylase from Actinoplanes utahensis NRRL12052. FR901379 acylase activity was found to be 250-fold higher in the recombinant Streptomyces lividans 1326 carrying the cloned gene than in the original Streptomyces sp. no. 6907 strain.

Natural fermentation products have long been studied as attractive targets for drug discovery due to their amazing diverse, complex chemical structures and biological activities. As such, a number of revolutionary drugs developed from natural fermentation products have contributed to global human health. To commercialize a drug derived from natural fermentation products, an effective chemical entity must be identified and thoroughly researched, and an effective manufacturing process to prepare a commercial supply must be developed. To construct such a manufacturing process for tacrolimus and micafungin, the following studies were conducted: first, we focused on controlling the production of the tacrolimus-related compound FR900525, a fermentation by-product of tacrolimus which was critical for quality assurance of the drug substance. FR900525 production was reduced by using a mutant strain which produced more pipecolic acid, the biosynthesis material of tacrolimus, than the original strain. Then, to optimize the fermentation process of FR901379, an intermediate of micafungin, a fed-batch culture was adopted to increase FR901379 productivity. Additionally, FULLZONE(TM) impeller was installed into the scaled-up fermenter, reducing the agitation-induced damage to the mycelium. As a result, the mycelial form changed from filamentous to pellet-shaped, and the air uptake rate during fermentation was drastically improved. Finally, we conducted screening for FR901379 acylase-producing microorganisms, as FR901379 acylase is necessary to manufacture micafungin. We were able to easily discover FR901379 acylase-producing microorganisms in soil samples using our novel, convenient screening method, which involves comparing the difference in antibiotic activity between FR901379 and its deacylated product.

In the presence of a Pd-catalyst and AgOCOCF3 as an oxidant, N-cinnamoylanilines (I), (IV) and (VI) afford 3-alkylidenoxindoles via an intramolecular C—H alkenylation reaction in moderate to good yields.