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The development of countercurrent chromatography (CCC) technology enabled to perform higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol (PEG) and inorganic salts (or dextrans). By eliminating solid support matrix, all analytes can be recovered from the coiled column after completing the separation. Recently, it has been found that droplets of biomolecules formed by liquid-liquid phase separation in cells closely relate to the transcription, regulation of signal transduction and formation of amyloids, etc. Meanwhile, although CCC is a separation technique based on liquid-liquid partitioning of analytes between two immiscible phases, the mechanism of separation could suggest some idea concerning the formation of biomolecule droplets in cells. This article describes the recent advances of the CCC apparatus, the coiled separation column, the choice of suitable two-phase solvent system and the application to separation and purification of bioactive macromolecules such as proteins and enzymes and also discusses the possibility of CCC as a tool to reveal new mechanical roles of biomolecule droplets in the cellular environments.

Catechol O-methyltransferase (COMT) is known as an important drug-target protein in the field of Parkinson's disease. All clinically approved COMT inhibitors bring a 5-substituted-3-nitrocatechol ring as a pharmacophore, and they bind to COMT with S-adenosylmethionine (SAM) and an Mg2+ ion to form a quaternary complex (COMT/SAM/Mg2+/inhibitor). However, structural information about such quaternary complexes is only available for a few inhibitors. Here, a new crystal structure of COMT complexed with nitecapone (5), SAM and Mg2+ is revealed. Comparison of the structures of these complexes indicates that conformation of the catechol binding pocket is almost constant regardless of structure of the inhibitors. The only restriction of the side chain of inhibitors (i.e., the substituent at the 5-position of 3-nitrocatechol) seems to be that it does not make steric repulsion with COMT. However, recent crystallographic and biochemical studies suggest that COMT is a flexible protein, and its conformational flexibility seems crucial for its catalytic process. Based on this information, implications of these quaternary inhibitor complexes were investigated. Met 40 in the α2α3-loop makes atomic contacts with SAM or S-adenosylhomocysteine and the 3-position of the catechol inhibitor. This interaction seems to play a critical role in the affinity of the inhibitor and to stabilize the COMT/SAM/Mg2+/nitrocatechol inhibitor complex by fixing the flexible α2α3-loop.

Microbiome analysis has attracted attention in recent years as a tool to monitor individual health status. In particular, gut microbiota is closely related to autoimmunity, and detailed study of gut microbiota may enable prediction of the health status of individuals.Here, we aimed to conduct a detailed study to identify gastric cancer-specific gut microbiota and compare the data between patients with gastric cancer and healthy individuals. Toward this, we extracted DNA from feces of patients with advanced gastric cancer enrolled in the observational/translational study, the DELIVER trial (JACCRO GC-08: UMIN000030850) which evaluated clinical outcomes of nivolumab and developed host-related biomarkers for nivolumab in 500 patients with advanced gastric cancer, and obtained their sequence data at an average of 5 Gbps using whole-genome shotgun sequencing. We then performed genus classification and functional pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG).We analyzed differences in bacterial composition using the data of 476 Japanese patients with advanced gastric cancer (median age 70y, median BMI 20.0, 71% male, 37.0% of patients with relapse, 55.5% with previous gastrectomy) and public database of 106 healthy cases. Bifidobacterium, Anaerostipes, and Parabacteroides were predominant in the healthy individuals, whereas Streptococcus, Lactobacillus, and Odoribacter were predominant in patients with advanced gastric cancer. Furthermore, the KEGG pathway analysis showed that butanoate and pyruvate metabolism were enriched in the healthy individuals, whereas factors such as ABC transporters and ribosomes were enriched in the patients with advanced gastric cancer. Clustering analysis broadly classified the patients with advanced gastric cancer and healthy individuals into two clusters, and the clustering using pathway data enabled clearer classification of the patients with advanced gastric cancer and healthy individuals than the clustering using flora analysis. Additionally, the healthy individuals had higher bacterial flora diversity compared to the patients with advanced gastric cancer.We thus successfully identified the molecular characteristic of microbial genera and functional pathways in the gut in patients with advanced gastric cancer. We conclude that fecal metagenomic shotgun sequencing analysis might be used to detect gastric cancer. Citation Format: Ryo Matoba, Hiroshi Iijima, Yasuhiro Sakamoto, Ryohei Kawabata, Atsushi Ishiguro, Yusuke Akamaru, Yosuke Kito, Hiroshi Yabusaki, Jin Matsuyama, Masazumi Takahashi, Akitaka Makiyama, Takahisa Suzuki, Masahiro Tsuda, Hisateru Yasui, Jun Hihara, Toru Masuzawa, Hiroyuki Okuda, Junji Kawada, Shuhei Suzuki, Hisato Kawakami, Takako Eguchi Nakajima, Kei Muro, Wataru Ichikawa, Masashi Fujii, Yu Sunakawa. Molecular characteristics of gut microbiota in patients with gastric cancer: The DELIVER trial (JACCRO GC-08) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5957.

COMT (Catechol-O-methyl transferase), an enzyme that metabolizes catechol, requires magnesium (Mg 2+ ) to maintain its activity. Low COMT activity causes insufficient 2-methoxyestradiol (2-ME), a biologically active metabolite from hydroxyestradiol, which leads to hypertensive disorders, including preeclampsia. Hypoestrogenism increases the risk of salt-sensitive hypertension (SSH). SSH and preeclampsia are risk factors for each other; however, the molecular mechanism of this interaction is unclear. We focused on the interactive effect of Mg 2+ insufficiency and genetic COMT deficiency on SSH using 2 strains of mice with genetically distinct COMT activity. In male mice, BL6 (C57BL/6J), a high-activity COMT strain, displayed unaltered blood pressure regardless of the Mg 2+ and salt levels in food; DBA (DBA/2J), a low-activity COMT strain, developed SSH under low Mg 2+ and high-salt conditions. COMT inhibition in C57BL/6J strain also induced SSH. Treatment with 2-ME ameliorated SSH in both models. The ATR1 (angiotensin II type 1 receptor)–STE20-SPAK (serine-proline alanine-rich kinase)–NCC (sodium chloride cotransporter) axis, molecules associated with sodium reabsorption in distal convoluted tubules, was activated in mice that developed SSH. In female DBA mice, ovariectomized mice displayed SSH under low Mg 2+ associated with activation of ATR1-SPAK-NCC axis; 2-ME inhibited all, whereas the blood pressure of sham mice was unaltered regardless of any intervention. Our findings revealed that Mg 2+ insufficiency exaggerated the low COMT activity and induced SSH via the ATR1-SPAK-NCC axis due to 2-ME insufficiency, suggesting a new pathophysiological role that links COMT/2-ME deficiency with hypertensive syndrome.

Catechol O-methyltransferase (COMT) is known as an important drug-target protein in the field of Parkinson's disease. All clinically approved COMT inhibitors bring a 5-substituted-3-nitrocatechol ring as a pharmacophore, and they bind to COMT with S-adenosylmethionine (SAM) and an Mg

Chondrocyte sheet transplantation is a novel and promising approach to treating patients who have cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration by autologous chondrocyte sheets has already been demonstrated in clinical research. In this study, the efficacy of polydactyly-derived chondrocyte sheets (PD sheets) as an allogeneic alternative to standard chondrocyte sheets was examined using an orthotopic xenogeneic transplantation model. In addition, the expression of genes and the secreted proteins in the PD sheets was analyzed using a microarray and a DNA aptamer array. The efficacy of PD sheets with respect to cartilage defects was assessed using histological scores, after which the expressions of genes and proteins exhibiting a correlation to efficacy were identified. Enrichment analysis of efficacy-correlated genes and proteins showed that they were associated with extracellular matrices, skeletal development, and angiogenesis. Eight genes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA) exhibited a positive correlation with the efficacy of PD sheets, and three genes (RARRES2, APOE, and PGF) showed a negative correlation for both transcriptomic and proteomic analyses. Among these, MIA, DKK1, and GREM1 involved in skeletal development pathways and ESM1 involved in the angiogenesis pathway exhibited a correlation between the amount of secretion and efficacy. These results suggest that these secreted factors may prove useful for predicting PD sheet efficacy and may therefore contribute to hyaline cartilage regeneration via PD sheets.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

To explore the structure-activity relationships of flavanonols on the inhibition of nitric oxide (NO) production in RAW 264.7 cells, we have prepared a series of synthetic flavanonols. In our previous study, the 2',3'-dihydroxyphenyl substructure was found to be the most potent B ring substructure among the flavanonols having 3,5,7-trihydroxychroman-4-one as the A/C ring. In this study, we examined the effect of diverse substitutions on the A ring of the 2-(2,3-dihydroxyphenyl)-3-hydroxychroman-4-one scaffold, i.e., by fixing the B ring to the 2',3'-dihydroxyphenyl substructure. Eighteen stereoisomers and 4 racemic mixtures were prepared, and their inhibitory potency on NO production in RAW 264.7 cells was tested. We observed higher inhibitory activity in the (2R,3R) stereoisomers than in the (2S,3S) stereoisomers. The presence of a hydroxy or a methoxy group at the 7-postiion enhanced the inhibitory potency, and the additional substitutions at the 6- or 8-position in the A ring increased potency and stereospecificity. A representative compound, (2R,3R)-2',3',7,8-tetrahydroxyflavanonol 5e, had an IC

Aim Stress during the perinatal period may result in failure to thrive in the newborn. A possible preventive effect of two kampo formulae, saikokaryukotsuboreito (SRB) and hachimijiogan (HJG) on the incidence of hind-leg paraparesis and mortality in newborns, including that due to maternal cannibalism, were examined in neurotoxin-treated rats. Methods Female Wistar/ST rats treated with SRB or HJG during pregnancy and the lactation period were separated from their pups for 6 h daily and then reunited. The pups were treated daily with a neurotoxin, l-β- N -oxalyl-α,β-diaminopropionic acid (l-β-ODAP), to cause neurolathyrism-like symptoms, a motor defect leading to hind-leg paraparesis. Results Although SRB and HJG treatment in mother rats did not affect the incidence of motor defect (paraparesis) in the pups, pup mortality was greatly and significantly decreased during the treatment period in the SRB group but not in the HJG group. Conclusion SRB treatment in mother rats is effective in decreasing pup mortality mainly due to cannibalism, thus suggesting its potential for anxiety disorder during the perinatal period in humans.

Bioactive natural products, habitually ingested with milk or its derivative nutrients, have been studied for their bioavailability. In this study, we investigated the effects of the co-administration of bovine milk-derived lactoferrin (bLF) and bioactive products, with a focus on catechol-O-methyltransferase (COMT), an enzyme in the catechol metabolism. bLF showed inhibitory activity on COMT in vitro, and acidic pretreatment of bLF enhanced its inhibitory activity. Moreover, partially digested products of bLF by pepsin retained inhibitory activity. Based on these results, bLF was co-administered with levodopa (L-DOPA), which is a catechol compound and a precursor of dopamine, and the effect of bLF on L-DOPA absorption and metabolism was investigated in a mouse model. The co-administration of L-DOPA and bLF alone showed no effect on the concentration of L-DOPA in plasma. However, with the additional administration of carbidopa, the concentration of L-DOPA was significantly enhanced. Furthermore, the ratio of L-DOPA/3-O-methyldopa significantly increased. On the other hand, casein, which is a major milk protein, was not effective. In addition, COMT activity in the intestines was lowered with bLF administration. We concluded that the co-administration of bLF and carbidopa enhances the concentration of L-DOPA.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

Lactoferrin (LF) is a well-known multifunctional protein. In this study, we report the inhibitory potency of bovine LF (bLF) on catechol-O-methyltransferase (COMT), which catalyzes methylation of catechol substrates. We found that bLF binds to and inhibits COMT using its N-terminal region. An N-terminal peptide fragment obtained from bLF by trypsin digestion showed a higher inhibitory activity than intact bLF. A synthetic fragment of the bLF N-terminal residues 6-50, with two pairs of disulfide bonds, also showed higher inhibitory activity than intact bLF. Enzyme kinetic studies proved that bLF did not compete with S-adenosylmethionine (the methyl donor substrate) as well as methyl acceptor substrates such as dihydroxybenzoic acid, (-)-epicatechin, norepinephrine, or l-3,4-dihydroxyphenylalanine. The inhibitory potency of bLF decreased against a COMT preparation pretreated with dithiothreitol, suggesting that the oxidation status of COMT is relevant to interaction with bLF. We further confirmed that COMT activity in the cell extracts form Caco-2 and HepG2 cells was inhibited by bLF and by the synthesized fragment. Enzyme kinetic study indicated that bLF functions as a non-competitive inhibitor by binding to an allosteric surface of COMT.

Essential tremor is a disorder that causes involuntary oscillations in patients both while they are engaged in actions and when maintaining a posture. Such patients face serious difficulties in performing daily living activities such as meal movement. We have been developing an electromyogram (EMG)-controlled exoskeleton to suppress tremors to support the movements of these patients. The problem is that the EMG signal of the patients is modulated by the tremor signal as multiplicative noise. In this paper, we proposed a novel signal processing method to demodulate patients’ EMG signals. We modelled the multiplicative tremor signal with a powered sine wave and the tremor signal in the EMG signal was removed by dividing the modelled tremor signal into the EMG signal. To evaluate the effectiveness of the demodulation, we applied the method to a real patient’s EMG signal, extracted from biceps brachii while performing an elbow flexion. We quantified the effect of the demodulation by root mean square error between two kinds of muscle torques, an estimated torque from the EMG signal and calculated torque from inverse dynamics based on the motion data. The proposed method succeeded in reducing the error by approximately 15–45% compared with using a low-pass filter, typical processing for additive noise, and showed its effectiveness in the demodulation of the patients’ EMG signal.

Zeolite L (LTL) can recognize cytochrome c (cyt c) in a model ternary protein mixture and completely separate it with 100% recovery from the mixture. LTL is a highly potential adsorbent for the purification of proteins.

Essential tremor is a disorder that causes involuntary oscillations in patients both while they are engaged in actions and when maintaining a posture. Such patients face serious difficulties in performing such daily living activities as eating, drinking, and writing. We have been developing an electromyogram-controlled exoskeleton to suppress tremors and support the eating movements of these patients. This exoskeleton is designed to suppress tremors and support voluntary movement at the elbow in terms of flexion and extension: movement of the elbow is essential in eating movements. In this study, we examined the effectiveness of our prototype exoskeleton at suppressing tremors. Our goal was to answer two questions: To what extent are the oscillations suppressed when wearing the exoskeleton? Is the exoskeleton able to suppress the oscillations sufficiently to allow eating movements? We were able to confirm experimentally that our exoskeleton can effectively suppress tremors to support eating movements.

We isolated flavonoids from herbal specimens from the Tibetan region (Sophora yunnanensis and Rhodiola sacra) that suppress nitric oxide (NO) production in macrophages stimulated by lipopolysaccharide and interferon-γ. The isolated flavonoids carry symmetric substitutions in the B ring (R3'=R5'). We analyzed the quantitative structure-activity relationship of the inhibitory activity by comparative molecular field analysis (CoMFA) using this series of flavonoids. Use of flavonoids with symmetrical substitutions in the B ring made it simpler to align molecules because it was not necessary to consider a huge number of combinations due to the B-ring conformation. The CoMFA model, whose cross-validated q2 value was 0.705, suggested the existence of a hydroxy group at the 5-position, the choice of the A/C-ring scaffold (chromane or chromene) and electrostatic field around the B ring are important for NO inhibitory activity. Flavonoids synthesized based on the CoMFA model exhibited significant inhibitory potential against NO production, validating the predictive capability of the CoMFA model.

Background According to EULAR recommendations, biologic DMARDs (bDMARDs) such as tumor necrosis factor inhibitor, tocilizumab (TCZ), and abatacept (ABT) are in parallel when prescribing to rheumatoid arthritis (RA) patients who have shown insufficient response to conventional synthetic DMARDs. However, most prediction studies of therapeutic response to bDMARDs using gene expression profiles were focused on a single bDMARD, and consideration of the results from the perspective of RA pathophysiology was insufficient. The aim of this study was to identify the specific molecular biological features predicting the therapeutic outcomes of three bDMARDs (infliximab [IFX], TCZ, and ABT) by studying blood gene expression signatures of patients before biologic treatment in a unified test platform. Methods RA patients who responded inadequately to methotrexate and were later commenced on any one of IFX (n = 140), TCZ (n = 38), or ABT (n = 31) as their first biologic between May 2007 and November 2011 were enrolled. Whole-blood gene expression data were obtained before biologic administration. Patients were categorized into remission (REM) and nonremission (NON-REM) groups according to CDAI at 6 months of biologic therapy. We employed Gene Set Enrichment Analysis (GSEA) to identify functional gene sets differentially expressed between these two groups for each biologic. Then, we compiled “signature scores” for these gene sets, and the prediction performances were assessed. Results GSEA showed that inflammasome genes were significantly upregulated with IFX in the NON-REM group compared with the REM group. With TCZ in the REM group, B-cell-specifically expressed genes were upregulated. RNA elongation, apoptosis-related, and NK-cell-specifically expressed genes were upregulated with ABT in the NON-REM group. Logistic regression analyses showed that “signature scores” of inflammasomes, B-cell-specifically expressed, and NK-cell-specifically expressed genes were significant, independently predictive factors for treatment outcome with IFX, TCZ, and ABT, respectively. The AUCs of ROC curves of these signature scores were 0.637, 0.796, and 0.768 for IFX, TCZ, and ABT, respectively. Conclusions We have identified original gene expression predictive signatures uniquely underlying the therapeutic effects of IFX, TCZ, and ABT. This is, to our knowledge, the first attempt to predict therapeutic effects of three drugs concomitantly using a unified gene expression test platform. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1052-8) contains supplementary material, which is available to authorized users.

A split belt treadmill for gait rehabilitation was developed to improve the symmetry of the stance phase time of patients with stroke. The system, which increases the stance phase time of the affected leg and then realizes a well-balanced gait, is divided into two components. First, the stance phases of the sound and affected legs were measured and presented visually in real time to the patient and physical therapist as biofeedback. Second, using stance phase biofeedback, the physical therapist sets two different velocities of treadmill belts for sound and affected legs. In an experiment, 11 patients with chronic stroke participated in a short-term intervention trial (20 gait cycles) of the developed treadmill system. Three of the five subjects who had lost balance between the stance phase of the sound leg and that of the affected one improved their gait balance in the intervention trial. In addition, one subject kept the well-balanced gait after the intervention.

The article describes the development of a gait phase time-based split-belt treadmill measurement system. Conventional methods of measuring gait phase, such as the foot switch and force plate, require significant preparation and are costly. In this article, we propose a simple, cheap, and accurate gait phase measurement system that utilizes only the treadmill motor current value. Comparison of this algorithm with conventional methods reveals that the proposed algorithm is as accurate as the foot switch. Moreover, the proposed algorithm can estimate stance phase within a 0.2 s error of the measured value of the force plate in most cases (four out of five healthy subjects). This accuracy is higher than that of the foot switch which is widely used in the clinical field.

Background: Golgi is a potential target for cancer treatment, but no inhibitor became an anticancer drug. Results: Using a unique bioinformatics approach, we identified a novel Golgi inhibitor, AMF-26, targeting Arf1 activation and possessing potent antitumor activity. Conclusion: AMF-26 is a promising new anticancer drug lead. Significance: Our data indicate that Arf1 activation is a promising target for cancer treatment., ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

We have been developing a robotic split-belt treadmill with different belt velocities and visual biofeedback of stance phase for the stroke patients to improve the symmetricity of their gait balance. In this treadmill system, a visual bio-feedback system of stance phase time of affected and unaffected legs is used. In this paper, a signal processing to eliminate spike and high frequency noises and oscillation of judge result of gait phase is discussed to indicate the stance phase time in real-time accurately. In addition, the effect of visual biofeedback of gait balance on their gait was analyzed in the determined biofeedback condition. Firstly, we conducted the gait experiment to determine the parameters in the signal processing such as cut-off frequency of low pass filter. In this experiment, the number of false judgment was calculated in normal gait, high cadence gait and simulated stroke gait. As a result, two parameters, cut-off frequency and the number of data to judge the gait phase, are set as 3.0 [Hz] and 10 respectively. In this condition, the gait phase of affected and unaffected legs are accurately displayed in front of stroke patient and physical therapist. Secondly, simulated stroke patients evaluated the effect of biofeedback of stance phase on the stance phase balance. As a result, the stance phase balance between affected and unaffected legs' stance phase was symmetrized by using visual biofeedback system.

Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).

Background/Aims: The plasma concentration of catecholamines and their metabolites generated by catechol-O-methyl transferase (COMT) were measured and their correlation with the progress of renal dysfunction was investigated in two distinctive animal models: a 5/6 nephrectomized Sprague-Dawley rat model and a 1/2 nephrectomized diabetic fatty Zucker rat model. Methods: A highly sensitive, high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection was employed to obtain values for the ratio [NMN]/([NE] + [NMN]), where [NE] represents the plasma concentration of norepinephrine and [NMN] represents the plasma concentration of normetanephrine. Results: The [NMN]/([NE] + [NMN]) ratio correlated with both the increase in blood urea nitrogen concentration and the decrease in creatinine clearance. Conclusion: The [NMN]/([NE] + [NMN]) ratio represents a quantitative indicator of the progress of renal dysfunction in the animal models. Regulation of COMT activity seemed to relate with the progress of renal dysfunction.

第2章 霞ケ浦を再生させる 飯島 博[NP0法人アサザ基金代表]

We isolated the glycolipids fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the activities of prokaryotic DNA polymerase I from Escherichia coli (E. coli) and cell growth of E. coli. The fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and purified SQDG inhibited these activities, however, purified MGDG and DGDG had no influence. In the tested strains of E. coli, SQDG inhibited the cell proliferation of the JM109 strain. It could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might have anti-bacterial activity.

To explore the structure-activity relationships on the inhibitory activity of flavanonols against nitric oxide (NO) production in inflammatory cells, we synthesized 19 flavanonols which shared a common 3,5,7-trihydroxychroman scaffold. A range of substitutions was included in the B ring in order to investigate the structure-activity relationship. We also succeeded in isolating stereoisomers from 16 of the flavanonols using chiral column chromatography. The inhibitory effects of these compounds on NO production were examined in RAW 264.7 cells (a murine macrophage-like cell line), which were activated by lipopolysaccharide (LPS). We only observed inhibitory activity against NO production in (2R,3R) stereoisomers, while the inhibitory activities of (2S,3S) stereoisomers were significantly weaker. We also evaluated the free radical scavenging potential of the flavanonols using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Each stereoisomer indicated the equivalent DPPH scavenging potential as expected. The radical scavenging activity was not correlated with the inhibitory activity against NO. The inhibition of NO production by flavanonols is stereospecific and cannot simply be explained by their radical scavenging activity. We propose the possible existence of a 'target' molecule for flavanonols which is involved in the production and/or regulation of NO in RAW 264.7 cells.

It was found that a class of sulfolipids known as sulfo-quinovosyl-acyl-glycerols (SQAGs) from ferns and algae are potent inhibitors of eukaryotic DNA polymerase alpha and beta and effective anti-neoplastic agents. In developing a procedure for the chemical synthesis of sulfolipids, many derivatives and stereoisomers of SQAGs have been obtained including sulfo-quinovosyl-monoacyl-glycerols (SQMGs) and sulfo-quinovosyl-diacyl-glycerols (SQDGs). This review describes studies on the structure-function relationship between synthetic SQAGs and DNA polymerase alpha and beta, and the relationship to cytotoxic activity. The major action was probably dependent on the fatty acid effect, which was reported previously, although each of the SQAGs was a much stronger inhibitor than just the fatty acid present in the SQAGs. The inhibitory effect could be influenced by the chain size of fatty acids in the SQAGs. The sulfonyl group in quinovose was also needed to inhibit the enzymes. Lineweaver-Burk plots of SQAGs indicated that DNA polymerase alpha was non-competitively inhibited, but the SQAGs were effective as antagonists of both the template-primer DNA-binding and the nucleotide substrate-binding of DNA polymerase beta. Based on these results, the molecular actions of SQAGs and drug design strategies for developing new anti-neoplastic agents were discussed.

We have investigated the thermal activation process of Mg dopant in Mg-doped GaN by secondary ion mass spectrometry (SIMS) and Hall measurement with the van der Pauw configuration. We have found a systematic relationship between the amount of the hydrogen dissociated from the Mg-doped GaN layer by thermal annealing and the electronic conductive properties of the layer, i.e., Hall mobility, Hall carrier density and resistivity of the layer. The hydrogen in the Mg-doped GaN layer has been classified into at least two different modes. The first mode involves relatively larger activation energy of the dissociation process from the Mg-doped GaN layer, and the dissociation energy from the Mg-doped GaN to the atmosphere is 0.8–1.5 eV. The second mode has the dissociation energy of 0.2–0.5 eV. The former mode may be associate with the hydrogen passivation of the Mg dopant in GaN and the latter one may be closely related to the passivation of the electrical compensation mechanism for the positive hole carriers in the Mg-doped GaN.

We discovered a new series of 4-phenoxyquinoline derivatives as potent and selective inhibitors of the platelet-derived growth factor receptor (PDGFr) tyrosine kinase. We researched the highly potent and selective inhibitors on the basis of both PDGFr and epidermal growth factor receptor (EGFr) inhibitory activity. First, we found a compound, Ki6783 (1), which inhibited PDGFr autophosphorylation at 0.13 microM, but it did not inhibit EGFr autophosphorylation at 100 microM. After extensive explorations, we found the two desired compounds, Ki6896 (2) and Ki6945 (3), which are substituted by benzoyl and benzamide at the 4-position of the phenoxy group on 4-phenoxyquinoline, respectively. These inhibitory activities were 0.31 and 0.050 microM, respectively, but neither of them inhibited EGFr autophosphorylation at 100 microM. We further investigated the profile of both compounds toward various tyrosine and serine/threonine kinases. The three compounds specifically inhibited PDGFr rather than the other kinases.

This study shows relationship between cognition of plants and animals and experience based on memory of childhood. Children did interview to their grandparents and parents to understand play, lifestyle and situation in waterside. It was view to give children opportunity of environmental education through having interview with familiar adults. Consequently sixty and seventy generations tended answer similar species, but the younger generation answered less than them as to rate. And it was showed that the plants have a closer relation to experience and cognition than animals. On the now, people are not the use of plants and animals in their life and most of adults feel the change of environment. This study will make this problem obvious to the way to get children interested in the change that adults experienced is needed for children.

The genus Dendrobium, one of the largest genera in Orchidaceae, exhibits enormous vegetative diversification. Such a situation has hindered the establishment of consistent classification systems. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we performed molecular phylogenetic analyses of 210 taxa. The results showed that many sections are not monophyletic. Most of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the results of convergence. Consequently, recircumscription of the infrageneric classification is required., It is always difficult to construct coherent classification systems for plant lineages having diverse morphological characters. The genus Dendrobium, one of the largest genera in the Orchidaceae, includes ∼1100 species, and enormous morphological diversification has hindered the establishment of consistent classification systems covering all major groups of this genus. Given the particular importance of species in Dendrobium section Dendrobium and allied groups as floriculture and crude drug genetic resources, there is an urgent need to establish a stable classification system. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we analysed the macromolecular characters of the group. Phylogenetic analyses of 210 taxa of Dendrobium were conducted on DNA sequences of internal transcribed spacer (ITS) regions of 18S–26S nuclear ribosomal DNA and the maturase-coding gene (matK) located in an intron of the plastid gene trnK using maximum parsimony and Bayesian methods. The parsimony and Bayesian analyses revealed 13 distinct clades in the group comprising section Dendrobium and its allied groups. Results also showed paraphyly or polyphyly of sections Amblyanthus, Aporum, Breviflores, Calcarifera, Crumenata, Dendrobium, Densiflora, Distichophyllae, Dolichocentrum, Holochrysa, Oxyglossum and Pedilonum. On the other hand, the monophyly of section Stachyobium was well supported. It was found that many of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the result of convergence. As such, many of the sections that have been recognized up to this point were found to not be monophyletic, so recircumscription of sections is required.

Essential tremor is a disorder that causes involuntary oscillations in patients both while they are engaged in actions and when maintaining a posture. Such patients face serious difficulties in performing such daily living activities as eating, drinking, and writing. We have been developing an electromyogram (EMG)-controlled exoskeleton to suppress tremors and to support the movements of these patients. In this paper, we proposed a novel signal processing method to demodulate the patients' EMG signal, which contains the voluntary movement signal and the tremor noise. This is a real-time signal processing method for multiple types of noise. From a technical point of view, this processing is essential because, at present, there are no real-time methods addressing the multiple noise sources. Also, from an application point of view, this method is essential for the accurate control of the exoskeleton depending on the patients' voluntary movement intention. We confirmed that the proposed method showed feasibility to demodulate the EMG signal which is modulated by the tremor signal.

アサザは絶滅危惧II類に分類される多年生の浮葉植物である.霞ケ浦は我が国におけるアサザの最大規模の自生地であった.しかし近年,霞ケ浦のアサザは急速に衰退していることがわかった.すなわち,1996年には34の局所個体群が確認され展葉範囲の総面積は99,497m2だったが,2000年には局所個体群数は14に,展葉範囲の総面積は10,081m2にまで減少していた.また1996年にみられた局所個体群のうち5つでは,異型花柱性植物としての健全な種子生産のために必要な複数の花型の開花が認められたが,2000年には複数の花型の開花が認められた局所個体群は1つのみになっており,この局所個体群以外ではほとんど種子が生産されていなかった.一方,1996年までは良好な種子生産がみられた局所個体群の近くの湖岸では,アサザの実生の出現が認められた.しかし,1999年にこれらの実生約2000個体に標識して生存を調査したところ,それら全てが死亡・消失しており,定着は認められなかった.アサザの幼株も見出されず,霞ケ浦のアサザ個体群では更新がまったく起こっていないことが示唆された.また2000年にも同じ場所で実生の調査を行ったところ,発芽量自体が激減していた.これは調査場所付近のアサザ局所個体群が1999年に消失し,種子が供給されなくなったためと考えられる.2000年に確認された実生は1998年以前に生産されて永続的土壌シードバンク中で生存していた種子が発芽したものと考えられ,同じことが毎年繰り返されれば,霞ケ浦のアサザの土壌シードバンクは無駄な発芽によって消費し尽くされることが予測された.

We developed laminin-derived AG73 peptide labeled liposomes for cancer specific gene therapy. AG73 peptide is well known as a ligand for syndecan-2 which is highly expressed in various cancer cells. Liposomes labeled with AG73 showed high efficient transfection efficiency in syndecan-2 overexpressing cells, and found that AG73 could be a superior molecule in the development of non-viral vector using liposomes for the gene delivery to syndecan-2 overexpressing cancer cells.

The concentration dependences of 133Cs and 19F chemical shifts for aqueous solutions of cesium perfluorooctanoate are analyzed by using a two-state model to ascertain the results in our previous st...