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Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring orientation of fluorescently labeled molecules within living cells in real-time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but due to difficulties in the rational three-dimensional design of protein connection, universal method for constrained tagging with fluorophore was not available. Here we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner and can target arbitrary biomolecules by combining with phage-display screening. As an initial test case of POLArIS, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISactwith FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens including whole bodies of developing embryos and animals, and also expressed in a cell-type/tissue specific manner.

Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.

The development of a technology that allows for analyzing microscopic spatial distribution and dynamics of small gaseous molecules such as inhalational anesthetics and odors would advance our understanding of its biological activities in living cells. However, direct observation of such small molecules by optical microscopy is still challenging. We propose a new pump–probe stimulated Raman scattering (SRS) microscopy method for studying the localization, transport and metabolism of gaseous molecules in a living organism in a label-free manner. A technical challenge is how to detect the Raman signal of a small amount of drug molecules that is typically overwhelmed by unwanted nonlinear background, including nonresonant background and coherent Raman scattering of surrounding cells and tissues. In particular, the latter Raman-induced background is essentially inevitable in most standard coherent anti-Stokes Raman scattering (CARS) and SRS systems. We show that these background issues can be overcome by introducing a new pump–probe, time-resolved SRS detection approach coupled with a pair of spectrally-focused, asymmetrically shaped probe pulses (T. Ito et al. APL Photonics (2018)). In the pump–probe scheme, a long-lived vibration of the targeted molecules can be efficiently probed after short-lived vibrations of other background molecules such as water and fatty acids become silent. This unique lifetime-selective signal detection provides a significantly enhanced vibrational signal contrast. As a proof-of-concept experiment, we demonstrate that the passive transport of inhalational anesthetic molecules from aqueous solution to adipose cells can be monitored by time-lapse SRS imaging.

Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified 'cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (α-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and α-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy.

Psychological distress and coping styles have been suggested to relate to altered function in the hypothalamic-pituitary-adrenal (HPA) axis, although there remains much to be understood about their relationships. High and low cortisol levels (or reactivity) both represent HPA axis dysfunction, with accumulated evidence suggesting that they are linked to different types of psychopathology. The dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test has been extensively used to identify HPA axis abnormalities in various psychiatric conditions including mood disorders; however, the possible associations of psychological distress and coping styles with HPA axis function have not been well documented using this test. Here, we examined the relationships of HPA axis reactivity as measured by the DEX/CRH test with subjectively perceived psychological distress and coping styles, both of which were assessed with self-report questionnaires, in 121 healthy volunteers. Subjects were divided into three groups by the cortisol suppression pattern, namely the incomplete-suppressors (DST-Cortisol ≥ 5 μg/dL or DEX/CRH-Cortisol ≥ 5 μg/dL), moderate-suppressors (DST-Cortisol < 5 μg/dL and 1 μg/dL ≤ DEX/CRH -Cortisol < 5 μg/dL), and enhanced-suppressors (DST-Cortisol < 5 μg/dL and DEX/CRH-Cortisol < 1 μg/dL). The enhanced-suppressors showed significantly higher scores in obsessive-compulsive, interpersonal sensitivity and anxiety symptoms and significantly more frequent use of avoidant coping strategy, compared to the other two groups. These results point to the important role of enhanced suppression of cortisol, or blunted cortisol reactivity, in non-clinical psychopathology such as avoidant coping strategy and greater psychological distress.

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 cm−1 inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.

Hot water extract of the aerial part of Yacon (Smallanthus sonchifolia, Compositae) showed potent free radical-scavenging activity and inhibitory effects on lipid peroxidation in rat brain homogenate. The most potent antioxidative activity focused on the 50% MeOH-eluted fraction on DIAION HP-20 column chromatography. The structure of the major component in the fraction was identified as 2,3,5-tricaffeoylaltraric acid (TCAA) based on spectroscopic evidence. The antioxidative activity of TCAA is superior to that of natural antioxidants such as (+/-)-catechin, alpha-tocopherol, and ellagic acid, and TCAA also showed selective maltase-inhibitory activity (IC(50) 49 microg/ml). As the hypoglycemic activity of Yacon extract was described in a previous report, the present results showing that the aerial part of Yacon has strong antioxidative activity may encourage its potential use as a food supplement to prevent type II diabetes.

Nine 2-arylbenzofurans isolated from Morus species were tested for their antimicrobial activities against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Micrococcus luteus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Among these compounds, chalcomoracin (a leaf phytoalexine of mulberry tree) exhibited considerable antibacterial activity against MRSAs (MICs 0.78 mug/ml).

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against B. subtilis (MIC = 1.56 microg/ml). Four xanthones, gerontoxanthone I, toxyloxanthone C, cudraxanthone S, and 1,3,7-trihydroxy-2-prenylxanthone, showed weak antibacterial activity against the bacterium (MICs = 3.13-6.25 microg/ml). These compounds also exhibited similar MIC values against methicillin-sensitive S. aureus, MRSAs, and Micrococcus luteus.

Axonal transport is the specialized and well-developed intracellular transport system for regulated and/or long-distance transport based on generalized cellular machineries. Among them, slow axonal transport conveys cytoplasmic proteins. The motor molecule, the nature of transporting complex and the transport regulation mechanism for slow transport are still unclarified. There has been a dispute regarding the nature of transporting complex of cytoskeletal proteins, polymer-sliding hypothesis versus subunit-transport theory. Recent data supporting the hypothesis of polymer sliding in cultured neurons only reconfirm the previously reported structure and this inference suffers from the lack of ultrastructural evidence and the direct relevance to the physiological slow transport phenomenon in vivo. Observation of the moving cytoskeletal proteins in vivo using transgenic mice or squid giant axons revealed that subunits do move in a microtubule-dependent manner, strongly indicating the involvement of microtubule-based motor kinesin. If the slow transport rate reflects the intermittent fast transport dependent on kinesin motor, we have to investigate the molecular constituents of the transporting complex in more detail and evaluate why the motor and cargo interaction is so unstable. This kind of weak and fluctuating interaction between various molecular pairs could not be detected by conventional techniques, thus necessitating the establishment of a new experimental system before approaching the molecular regulation problem.

Nineteen flavonoids isolated from licorice (Glycyrrhiza glabra, G. inflata and G. uralensis) were tested for their antimicrobial activities against methicillin sensitive Staphylococcus aureus, methicillin resistant S. aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

The kinesin superfamily motor protein KIF1B has been shown to transport mitochondria. Here, we describe an isoform of KIF1B, KIF1Bβ, that is distinct from KIF1B in its cargo binding domain. KIF1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the β isoform. The KIF1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.

Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.

Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.

Deletion of the synapsin I genes, encoding one of the major groups of proteins on synaptic vesicles, in mice causes late onset epileptic seizures and enhanced experimental temporal lobe epilepsy. However, mice lacking synapsin I maintain normal excitatory synaptic transmission and modulation but for an enhancement of paired-pulse facilitation. To elucidate the cellular basis for epilepsy in mutants, we examined whether the inhibitory synapses in the hippocampus from mutant mice are intact by electrophysiological and morphological means. In the cultured hippocampal synapses from mutant mice, repeated application of a hypertonic solution significantly suppressed the subsequent transmitter release, associated with an accelerated vesicle replenishing time at the inhibitory synapses, compared with the excitatory synapses. In the mutants, morphologically identifiable synaptic vesicles failed to accumulate after application of a hypertonic solution at the inhibitory preterminals but not at the excitatory preterminals. In the CA3 pyramidal cells in hippocampal slices from mutant mice, inhibitory postsynaptic currents evoked by direct electrical stimulation of the interneuron in the striatum oriens were characterized by reduced quantal content compared with those in wild type. We conclude that synapsin I contributes to the anchoring of synaptic vesicles, thereby minimizing transmitter depletion at the inhibitory synapses. This may explain, at least in part, the epileptic seizures occurring in the synapsin I mutant mice.

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron– specific microtubule plus end–directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769–780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.

Eight 2-arylbenzofurans and an isoflavone isolated from medicinal plants were tested for their antimicrobial activities against vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, six hydrophobic 2-arylbenzofurans (log P = 4.4-8.7) exhibited considerable antibacterial activity against five VRE strains(VanA-, VanB-, and VanC-phenotypes) (MICs = 3.13-6.25 microg/mL). Five compounds also showed antibacterial activity against ten MRSA strains (MIC80 = 3.13 microg/mL).

The tau gene encodes a protein (Tau) that is a major neuronal microtubule-associated protein localized mostly in axons. It has microtubule-binding and tubulin-polymerizing activity in vitro and is thought to make short crossbridges between axonal microtubules. Further, tau-transfected non-neuronal cells extend long axon-like processes in which microtubule bundles resembling those in axons are formed. In contrast, tau antisense oligonucleotides selectively suppress axonal elongation in cultured neurons. Thus tau is thought to be essential for neuronal cell morphogenesis, especially axonal elongation and maintenance. To test this hypothesis, we used gene targeting to produce mice lacking the tau gene. We show that the nervous system of tau-deficient mice appears to be normal immunohistologically. Furthermore, axonal elongation is not affected in cultured neurons. But in some small-calibre axons, microtubule stability is decreased and microtubule organization is significantly changed. We observed an increase in microtubule-associated protein 1A which may compensate for the functions of tau in large-calibre axons. Our results argue against the suggested role of tau in axonal elongation but confirm that it is crucial in the stabilization and organization of axonal microtubules in a certain type of axon.

Syringaresinol isolated from Epimedium koreanum NAKA1 and Magnolia officinalis REHD. et WILS. was subjected to optical resolution by chiral HPLC to give (+)- and (-)-enantiomers. The two syringaresinol enantiomers, as well as a mixture of their glucosides, showed dose-dependent neuritogenesis in a concentration range from 0.24 to 24 microM in PC12h cells.

Inhalational anesthetic is important clinically, especially for surgical operations. However, the molecular mechanism of their action has not been completely clarified, because these anesthetic molecules are small, and hence, difficult in labeling in cells. To visualize their distribution in the cell and to examine in-vivo behavior of such a halogenated anesthetic, label-free biomedical imaging with molecular specificity is required.

Triamino-substituted 1, 3, 5-triazine derivatives were synthesized and tested for inhibitory activities against the aromatase of human lacental microsomes and the cytochrome P450 side chain cleavage of cholesterol (P450SCC) of pig adrenal mitochondria. The compounds having imidazolyl and tertiary amino groups as substituents in the 1, 3, 5-triazine ring showed significant aromatase-inhibitory activity. Among them, compounds 17, 23, 26, 27 and 28 were more active than the reference compound, CGS 16949A. The inhibitory activities of these compounds against P450SCC were much weaker than their aromatase-inhibitory activities. These compounds may be regarded as selective aromatase inhibitors.

Single-pulse heterodyne CARS (coherent anti-Stokes Raman scattering) detection scheme using shaped fem-tosecond pulses is one of the most sophisticated approach for managing the problem of non-resonant backgrounddisturbance in CARS measurement. However, with the signal processing method conducted in the originalreport, 1–3 we found that background suppression and resonant peak extraction were sometimes dicult andincomplete. We discuss the reason of t his unsuccessful signal processing and propose an improved method forsignal extraction realizing the better quality of extracted spectra.Keywords: coherent anti-Stokes Raman scattering, matched “lter, heterodyne detection, chloroform 1. INTRODUCTION It is widely accepted that CARS spectrometry in general suers from the non-resonant background, which oftenaggravates the visibility of the resonant CARS signal. Alt hough there have existed many early eorts aiming tosuppress the non-resonant signal, these kinds of attempts have not been easy tasks. To overcome this situation,Oron et al. (2002) originally reported a single-pulse coherently controlled CARS spectroscopy.

Attachment to the substrate is essential for both survival and differentiation of various kinds of cells, such as neurons and epithelial cells. We recently found a small synthetic molecule, adhesamine, which boosts adhesion and growth of mammalian cells. In the present study, we applied adhesamine to primary cultured hippocampal neuronal cells and compared its effects with those of PLL (poly-L-lysine), which is widely used as a substrate for cell cultures. Neurons grown on adhesamine-coated coverslips survived for up to 1 month without a feeder layer of glial cells, and had greater viability than cells grown on PLL-coated coverslips. Morphological analysis revealed that neurons cultured with adhesamine exhibited earlier differentiation, i.e. earlier axonal outgrowth and dendritic maturation with enhanced neurite branching, than neurons cultured with PLL. Synaptic formation and postsynaptic responses were evident as early as 4 days in cells cultured with adhesamine. Acceleration of differentiation is mediated by earlier activation of the signalling pathways from heparan sulfate in the extracellular matrix to both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). Improved survival rates and accelerated maturation of neurons exposed to adhesamine suggest that this completely synthetic molecule may be a useful reagent for culturing neuronal cells.

Previous functional neuroimaging studies have demonstrated that patients with schizophrenia and those with schizotypal personality disorder (SPD) show reduced laterality, or relative right hemispheric dominance, during the performance of cognitive activation tasks; however, neuroimaging studies looking at non-clinical schizotypy have been few. We have recently reported that schizotypal traits at a non-clinical level are associated with right prefrontal dominance during a letter version of the verbal fluency task (VFT), but it is unknown whether such relationship between schizotypy and functional laterality would be observed across various cognitive tasks. Here we examined the relationships of schizotypal traits as measured by the Schizotypal Personality Questionnaire (SPQ) in healthy adults with hemispheric lateralization of prefrontal activation during letter and category VFTs, using near-infrared spectroscopy. Thirty-two participants were divided into high- (n=16) and low- (n=16) SPQ groups by the median split of the total SPQ score. The high-SPQ group, but not low-SPQ group, showed significantly right-greater-than-left asymmetry of prefrontal activation during letter VFT, whereas such pronounced hemispheric asymmetry in relation to schizotypy was not found during category VFT. These results indicate that non-clinical schizotypy is related to right prefrontal preference during the letter version of VFT in particular, suggesting that the association between schizotypal traits and functional laterality may vary depending on cognitive activation tasks.

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a−/− mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a−/− growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a−/− cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.

Licorice is the most used crude drug in Kampo medicines (traditional Chinese medicines modified in Japan). The extract of the medicinal plant is also used as the basis of anti-ulcer medicines for treatment of peptic ulcer. Among the chemical constituents of the plant, glabridin and glabrene (components of Glycyrrhiza glabra), licochalcone A (G. inflata), licoricidin and licoisoflavone B (G. uralensis) exhibited inhibitory activity against the growth of Helicobacter pylori in vitro. These flavonoids also showed anti-H. pylori activity against a clarithromycin (CLAR) and amoxicillin (AMOX)-resistant strain. We also investigated the methanol extract of G. uralensis. From the extract, three new isoflavonoids (3-arylcoumarin, pterocarpan, and isoflavan) with a pyran ring, gancaonols A[bond]C, were isolated together with 15 known flavonoids. Among these compounds, vestitol, licoricone, 1-methoxyphaseollidin and gancaonol C exhibited anti-H. pylori activity against the CLAR and AMOX-resistant strain as well as four CLAR (AMOX)-sensitive strains. Glycyrin, formononetin, isolicoflavonol, glyasperin D, 6,8-diprenylorobol, gancaonin I, dihydrolicoisoflavone A, and gancaonol B possessed weaker anti-H. pylori activity. These compounds may be useful chemopreventive agents for peptic ulcer or gastric cancer in H. pylori-infected individuals.

Vigorous investigation has finally begun to shed light on the cargo problem of the microtubule-dependent motors, kinesin and dynein superfamily proteins. Biochemical observations have suggested that the potential cargoes of certain populations of motor proteins seem to be in vesicle-form, each vesicle possessing specific functional marker molecules. In addition to the close relationship between microtubule-dependent motors and cargoes in vesicle-form, kinesin has also been highlighted as an apparent driving force for another cargo in non-vesicle-form, cytoplasmic protein. On the basis of new biophysical and cell-biological evidence, the controversy over the movement of cytoplasmic cargoes has entered a new phase.

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

Triamino-substituted 1, 3, 5-triazine derivatives were synthesized and tested for inhibitory activities against the aromatase of human lacental microsomes and the cytochrome P450 side chain cleavage of cholesterol (P450SCC) of pig adrenal mitochondria. The compounds having imidazolyl and tertiary amino groups as substituents in the 1, 3, 5-triazine ring showed significant aromatase-inhibitory activity. Among them, compounds 17, 23, 26, 27 and 28 were more active than the reference compound, CGS 16949A. The inhibitory activities of these compounds against P450SCC were much weaker than their aromatase-inhibitory activities. These compounds may be regarded as selective aromatase inhibitors.

Introduction Altered levels of phenylalanine and its metabolites in blood and cerebrospinal fluid have previously been reported in schizophrenia. This study attempted to examine whether phenylalanine kinetics is altered in schizophrenia using the 13 C-phenylalanine breath test ( 13 C-PBT). Methods Subjects were 20 patients with schizophrenia and the same number of controls. 13 C-phenylalanine was administered and then 13 CO 2 concentration in breath was monitored for 120 minutes. The Δ 13 CO 2 at each collecting time, the maximal Δ 13 CO 2 (C max ), the time to reach C max (T max ), the area under the curve of time course of Δ 13 CO 2 (AUC), the cumulative recovery rate (CRR) at each collecting time of the 13 C-PBT were calculated for each subject. Results Body weight (BW) and diagnostic status were significant predictors for C max . BW, age and diagnostic status were significant predictors for AUC and CRR at 120 minutes (CRR 0-120 ). A repeated measures ANCOVA controlling for age and BW revealed a different pattern of change in CRR over time between the patients and controls and that Δ 13 CO 2 in schizophrenia were lower than that in healthy control at all sampling point during 120 min, with an overall significant differences between healthy control and schizophrenia. The ANCOVA controlling for age and BW, showed that C max , AUC and CRR 0-120 were significantly lower in schizophrenics than in controls. Conclusions Our data indicate the different change of Δ 13 CO 2 and CRR over time and the decreased C max , AUC and CRR 0-120 of 13 C-PBT in schizophrenia patients compared to healthy controls, suggesting the altered phenylalanine kinetics in schizophrenia.

In axons, cytoskeletal constituents move by slow transport. However, it remains controversial whether axonal neurofilaments are dynamic structures in which only subunits are transported or whether filaments assemble in the proximal axon and are transported intact as polymers to the axon terminus. To investigate the form neurofilament proteins take during transport, neurons of transgenic mice lacking axonal neurofilaments were infected with a recombinant adenoviral vector encoding epitope-tagged neurofilament M. Confocal and electron microscopy revealed that the virally encoded neurofilament M was transported in unpolymerized form along axonal microtubules. Thus, neurofilament proteins are probably transported as subunits or small oligomers along microtubules, which are major routes for slow axonal transport.

Halogenated volatile anesthetics are frequently used for inhaled anesthesia in clinical practice. No appropriate biological method has been available for visualizing their localization in action. Therefore, despite their frequent use, the mechanism of action of these drugs has not been fully investigated. We measured coherent anti-Stokes Raman scattering (CARS) spectra of sevoflurane and isoflurane, two of the most representative volatile anesthetics, and determined the low-frequency vibrational modes without nonresonant background disturbance. Molecular dynamics calculations predict that these modes are associated with multiple halogen atoms. Because halogen atoms rarely appear in biological compounds, the entire spectral landscape of these modes is expected to be a good marker for investigating the spatial localization of these drugs within the intracellular environment. Using live squid giant axons, we could detect the unique CARS spectra of sevoflurane for the first time in a biological setting.

Four antitumor active condurangoglycosides-B0, -D0, and 20-O-methyl-and 20-iso-O-methyl-condurangoglycoside-D0 were isolated from Condurango Cortex. Their structures were established by chemical and spectral data. Their antitumor activities and toxicities are described.

In order to confirm the structure of condurangogenin B, which is the aglycone of antitumor-active condurango glycoside (CG) B0, the hypoiodite reaction of condurangogenin C 3-acetate, was carried out. A mixture of condurangogenin C 3-acetate, Pb (OAc)4, I2, CaCO3, and diethyl ether was irradiated under nitrogen using a tungsten lamp. HPLC separation of the reaction products gave three compounds, P-1, P-2, and P-3. The structures of these compounds were determined from the spectroscopic data. The PMR and mass spectra of P-1 were essentially identical with those of condurangogenin B 3-acetate. Thus, the ester linkage positions (11-cinnamoyl, 12-acetyl) and the ketal structure of conduragogenin B were confirmed. As CGD0, 20-O-methyl-CGD0, and 20-iso-O-methyl-CGD0 could be converted chemically to CGB0, these glycosides have the same positions of ester linkages as CGB0.

A new polyoxypregnane derivative, stephanthraniline A, was isolated from the stem of Stephanotis japonica MAKINO (Asclepiadaceae), and its structure was determined.

In addition to mulberrofuran C, a new 2-arylbenzofuran derivative, six flavonoid derivatives, cyclomorusin, morusin, kuwanon C, E, G and H, as well as a known 2-arylbenzofuran derivative, chalcomoracin, were isolated from extracts of root bark of the cultivated mulberry tree (a variety of Morus bombycis K OIDZUMI). The structure of mulberrofuran C was shown to be VIII on the basis of spectral data. It is regarded biogenetically as a Diels-Alder adduct of a chalcone derivative and a dehydroprenyl 2-arylbenzofuran derivative. Intravenous injection of mulberrofuran C produced significant hypotension in rabbit.