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  • Articles  (6,813)
  • Oxford University Press
  • 2010-2014  (1,661)
  • 1980-1984  (2,588)
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  • 1950-1954  (334)
  • Chemistry and Pharmacology  (5,569)
  • Electrical Engineering, Measurement and Control Technology  (1,244)
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  • Articles  (6,813)
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  • 1
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    Affinity maturation of a CDR3-grafted VHH using in silico analysis and surface plasmon resonance (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al. , Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971–1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ~20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.
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    Topics: Biology , Chemistry and Pharmacology
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  • 2
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    Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ~30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.
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  • 3
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    TRIM proteins as trim tabs for the homoeostasis (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: The tripartite motif (TRIM) or RBCC proteins are characterized by the TRIM composed of a RING finger, B-box and coiled-coil domains. TRIM proteins often play roles in the post-translational protein modification, including ubiquitylation and other ubiquitin-like modifications. Evidence has accumulated in regard to the contribution of TRIM proteins to diverse cellular processes, including such as cell cycle progression, apoptosis, immunity and transcriptional regulation. In particular, some of the TRIM proteins have been characterized to exert oncogenic or tumour suppressor-like functions depending on the context. A recent report by Inoue and his colleagues has revealed that Terf/TRIM17 stimulates the degradation of a kinetochore protein ZWINT and regulates the proliferation of breast cancer cells. Terf has also been paid attention as a factor promoting neuronal apoptosis, by degrading a Bcl2-like anti-apoptotic protein Mcl-1. Like aircraft trim tabs, TRIM proteins trim the balance of homoeostasis by modulating various biological pathways through protein–protein interactions.
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  • 4
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    Inhibition of DNA binding of MCM2-7 complex by phosphorylation with cyclin-dependent kinases (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo . There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.
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  • 5
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    Effects of alcohols on the stability and low-frequency local motions that control the slow changes in structural dynamics of ferrocytochrome c (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: To determine the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact states of proteins, we have studied the effects of alcohols on structural fluctuation of M80-containing -loop by measuring the rate of thermally driven CO dissociation from a natively folded carbonmonoxycytochrome c under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3°-butanol, 2,2,2-trifluoroethanol). As alcohol is increased, the rate coefficient of CO dissociation ( k diss ) first decreases in subdenaturing region and then increases on going from subdenaturing to denaturing milieu. This decrease in k diss is more for 2,2,2-trifluroethanol and 1-propanol and least for methanol, indicating that the first phase of motional constraint is due to the hydrophobicity of alcohols and intramolecular protein cross-linking effect of alcohols, which results in conformational entropy loss of protein. The thermal denaturation midpoint for ferrocytochrome c decreases with increase in alcohol, indicating that alcohol decrease the global stability of protein. The stabilization free energy ( G ) in alcohols’ solution was calculated from the slope of the Wyman–Tanford plot and water activity. The m -values obtained from the slope of G versus alcohols plot were found to be more negative for longer and linear chain alcohols, indicating destabilization of proteins by alcohols through disturbance of hydrophobic interactions and hydrogen bonding.
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  • 6
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    Linear ubiquitination-mediated NF-{kappa}B regulation and its related disorders (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-B (NF-B) signalling. NF-B is crucial for inflammatory and immune responses, and aberrant NF-B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson’s disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-B activation pathway regulation by LUBAC.
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  • 7
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    Different circadian expression of major matrix-related genes in various types of cartilage: modulation by light-dark conditions (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light–dark and constant darkness conditions. Type II collagen and aggrecan genes—along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2—showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light–dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.
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  • 8
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    Association of epigenetic alterations in the human C7orf24 gene with the aberrant gene expression in malignant cells (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Human chromosome 7 open reading frame 24 (C7orf24)/-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.
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  • 9
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    Identification and characterization of interactions between abscisic acid and human heat shock protein 70 family members (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
    Description: Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA–HSP70 interactions yielded K d s in the 20–60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA–HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.
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  • 10
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    RETRACTION of: Down-regulation of protein tyrosine phosphatase gene expression in lactating mouse mammary gland (2013)
    Oxford University Press
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    Publication Date: 2013-10-02
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  • 11
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    Microscopy in this issue (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 12
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    Yeast and fungal cell-wall polysaccharides can self-assemble in vitro into an ultrastructure resembling in vivo yeast cell walls (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: Polysaccharides account for more than 90% of the content of fungal cell walls, but the mechanism underlying the formation of the architecture of the cell walls, which consist of microfibrils embedded in an amorphous wall matrix, remains unknown. We used electron microscopy to investigate ten different fungal cell-wall polysaccharides to determine whether they could self-assemble into the fibrillar or amorphous component of fungal cell walls in a test tube without enzymes. The ultrastructures formed by precipitating β-1,3-glucan and β-1,6-glucan are different depending on the existence of branching in the molecule. Linear β-1,3-glucan and linear β-1,6-glucan precipitate into a fibrillar ultrastructure. Branched β-1,6-glucan, mannan and glycogen precipitates are amorphous. Branched β-1,3-glucan forms a fibrillar plus amorphous ultrastructure. Self-assembly among combinations of different linear and branched cell-wall polysaccharides results in an ultrastructure that resembles that of a yeast cell wall, which suggests that self-assembly of polysaccharides may participate in the development of the three-dimensional architecture of the yeast cell wall.
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  • 13
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    Endocytic mechanism of transferrin-conjugated nanoparticles and the effects of their size and ligand number on the efficiency of drug delivery (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: When considering drug delivery, the amount of drug that can be carried at a particular time and how the drug is incorporated efficiently into cells are important parameters. Transferrin (Tf)-conjugated nanocarriers have been used for the targeted delivery of drugs to cancer cells due to the availability of receptor-mediated clathrin-dependent endocytosis. In general, however, endocytosis seems to differ according to the size and shape of carriers. Large substances are generally internalized into cells by phagocytosis. We studied the internalization mechanism of Tf-conjugated nanoparticles (diameter, 522 nm). Tf-conjugated polystyrene particles were incorporated into cells by receptor-mediated endocytosis with large clathrin-coated vesicles even though their diameter was 〉500 nm and despite that fact that clathrin-coated vesicles have a diameter of 100 nm. This finding suggests that signals for internalization generated by stimulated Tf receptors (TfRs) activate clathrin-mediated endocytosis preferentially. Whether these larger particles could deliver drugs more efficiently than smaller particles was then examined. The toxicity of larger Tf-conjugated biodegradable nanoparticles (poly(lactic-co-glycolic acid)) encapsulating doxorubicin (diameter, 216 ± 38 nm) was appreciably dependent on the number of Tf molecules conjugated on a particle and the number of TfRs expressed on the cell membrane. Larger Tf-conjugated particles delivered drugs to cancer cells expressing many TfRs more selectively than their smaller counterparts (diameter, 56 ± 9 nm) if they were decorated with an appropriate number of Tf molecules.
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  • 14
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    Imaging and diffraction of protein crystallization using TEM (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: Structural biology relies on good-quality protein crystals in order for structure determination. Many factors affect the growth process of a protein crystal including the way it nucleates and the types of damage and contamination during its growth. Although the nucleation process and quality of a crystal is vital to structure determination, they are both under-studied areas of research. Our research begins to explore ways of measuring the quality of protein crystals, using TEM, thus overcoming the problems associated with viewing wet specimens in a vacuum. Our current understanding of nucleation is that it is a two-step mechanism involving the formation of nuclei from dense liquid clusters; however; it is still unclear whether nuclei first start as amorphous aggregates or as crystalline lattices. Potentially, electron diffraction may be capable of uncovering this process. Using TEM imaging and diffraction of lysozyme as a model protein crystal, we report the internal two-dimensional strain and the density of crystallites in a protein crystal, at a resolution never seen before. The TEM diffraction shows unique features of crystal mosaicity that can be directly correlated to TEM images.
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  • 15
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    Micro-CT observations of the 3D distribution of calcium oxalate crystals in cotyledons during maturation and germination in Lotus miyakojimae seeds (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins; however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels.
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  • 16
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    Optimal accelerating voltage for HRTEM imaging of zeolite (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: The electron irradiation damage of MFI-type zeolite was estimated under various accelerating voltages of 100, 200 and 300 kV from successively captured high-resolution transmission electron microscopy (HRTEM) images. To determine the optimal accelerating voltage for HRTEM imaging of electron-sensitive MFI zeolite, the critical dose was estimated from the disappearance of a specific fast Fourier transform spot calculated from experimental images. Based only on the electron dose, a higher voltage was more advantageous. However, taking into account the minimum dose for imaging with a CCD camera, the optimal accelerating voltage for imaging MFI zeolite was 200 kV. The minimum dose for image detection with a CCD camera was surmised from the output/input signal ratio dependence on the accelerating voltage and the contrast range in simulated HRTEM images.
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  • 17
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    Electron holography of magnetic field generated by a magnetic recording head (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: The magnetic field generated by a magnetic recording head is evaluated using electron holography. A magnetic recording head, which is connected to an electric current source, is set on the specimen holder of a transmission electron microscope. Reconstructed phase images of the region around the magnetic pole show the change in the magnetic field distribution corresponding to the electric current applied to the coil of the head. A simulation of the magnetic field, which is conducted using the finite element method, reveals good agreement with the experimental observations.
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  • 18
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    A new grating X-ray spectrometer for 2-4 keV enabling a separate observation of In-L{beta} and Sn-L{alpha} emissions of indium tin oxide (2013)
    Oxford University Press
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    Publication Date: 2013-06-12
    Description: A new multilayer-coated varied line-spaced grating, JS4000, was fabricated and tested for extending the upper limit of a grating X-ray spectrometer for electron microscopy. This grating was designed for 2–3.8 keV at a grazing incidence angle of 1.35°. It was revealed that this new multilayer structure enables us to take soft-X-ray emission spectra continuously from 1.5 to 4.3 keV at the same optical setting. The full-width at half maximum of Te-L α1,2 (3.8 keV) emission peak was 27 eV. This spectrometer was applied to indium tin oxide particles and clearly resolved Sn-L α (3444 eV) and In-L β1 (3487 eV) peaks, which could not be resolved by a widely used energy-dispersive X-ray spectrometer.
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  • 19
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    Microscopy in this issue (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
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  • 20
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    Microscopy Hacks: development of various techniques to assist quantitative nanoanalysis and advanced electron microscopy (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: For development of advanced materials, characterization using a scanning transmission electron microscope (STEM) including analysis via X-ray energy-dispersive spectrometry and electron energy-loss spectrometry is essential. Recent advances in aberration-corrected instruments have offered large-scale data acquisition at a high resolution for limited acquisition times both in imaging and in analysis. Further advanced procedures are required to analyze such large-scale datasets more efficiently including quantification. In addition, more simplified tuning procedures are crucial to the best possible resolution in the latest aberration-corrected instruments. In this review article, several approaches to perform advanced electron microscopy, which the author has been developing with his colleague, are described as ‘Microscopy Hacks’. These are (i) quantification and elemental/chemical-imaging procedures, (ii) advanced statistical approaches to handle large-scale datasets and (iii) instrument characterization and tuning procedures including the latest development of an ad hoc autotuning procedure for aberration-corrected STEM imaging.
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  • 21
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    A new type of molybdenum oxide crystal encapsulated inside a single-walled carbon nanotube (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: The crystal structure of a new type of molybdenum oxide crystal encapsulated in a single-walled carbon nanotube (CNT) was examined via diffraction and spectroscopic techniques using both X-rays and electron beams. This new type of molybdenum oxide crystal has a chemical bonding state of MoO 3 , as confirmed by X-ray absorption spectroscopy, and the MoO 3 units exhibit axial symmetry, as clarified by electron diffraction from bundled and individual CNTs encapsulating the crystal. To obtain three-dimensional information on the structure, a cross-sectional sample was prepared using a conventional dimple and ion-mill method. High-resolution transmission electron microscopy images exhibit ring-like shapes that originated from the arrangement of the MoO 3 units inside the CNTs, as observed along the tube axis. The units are spaced 0.36 nm from each other in a ring arrangement and the distance between each ring is 0.391 nm.
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  • 22
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    Transmission electron microtomography in soft materials (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: This review summarizes the recent advances in three-dimensional (3D) imaging techniques and their application to polymer nanostructures, for example, microphase-separated structures of block copolymers. We place particular emphasis on the method of transmission electron microtomography (electron tomography for short; hereafter abbreviated as ET). As a result of recent developments in ET, truly quantitative 3D images of polymer nanostructures can now be obtained with subnanometer resolution. The introduction of scanning optics in ET has made it possible to obtain large amounts of 3D data from micrometer-thick polymer specimens by using conventional electron microscopes at a relatively low accelerating voltage, 200 kV. Thus, ET covers structures over a wide range of thicknesses, from a few nanometers to several hundred nanometers, which corresponds to quite an important spatial range for hierarchical polymer nanostructures. ET provides clear 3D images and a wide range of new structural information that cannot be obtained using other methods. Information traditionally derived from conventional microscopy or scattering methods can be directly acquired from 3D volume data. ET is a versatile technique that is not restricted to only polymer applications; it can also be used as a powerful characterization tool in energy applications such as fuel cells.
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    Performances of an 80-200 kV microscope employing a cold-FEG and an aberration-corrected objective lens (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: The performances of a newly developed 80–200 kV cold field emission gun (CFEG) transmission electron microscope (TEM) integrating a spherical aberration corrector for a TEM image-forming lens have been evaluated. To begin, we show that the stability of both emission and probe currents makes use of this new CFEG much friendlier. The energy spread of electrons emitted from the CFEG has been measured as a function of emission current and shows a very last 0.26 eV energy resolution at 200 kV and even 0.23 eV at 80 kV. The combination of the CFEG and the CEOS™ aberration corrector, associated with enhanced mechanical and electrical stabilities of this new microscope, allows reaching an information transfer below 75 pm at 200 and 80 pm at 80 kV. This unseen resolution at 200 kV has allowed us to study the structure of CoPt nanoparticles by observing direct images of their atomic arrangement along the high indexes zone axis. We have evidenced the presence of defects in these nanostructures that are not parallel to the electron beam. The precise stoichiometry of two iron oxides, FeO and Fe 2 O 3 , has been determined from an analysis of iron valence state that was obtained from a direct analysis of EELS fine structures spectrum of the two oxides.
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    Multi-reference-based multiple alignment statistics enables accurate protein-particle pickup from noisy images (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: Data mining from noisy data/images is one of the most important themes in modern science and technology. Statistical image processing is a promising technique for analysing such data. Automation of particle pickup from noisy electron micrographs is essential, especially when improvement of the resolution of single particle analysis requires a huge number of particle images. For such a purpose, reference-based matching using primary three-dimensional (3D) model projections is mainly adopted. In the matching, however, the highest peaks of the correlation may not accurately indicate particles when the image is very noisy. In contrast, the density and the heights of the peaks should reflect the probability distribution of the particles. To statistically determine the particle positions from the peak distributions, we have developed a density-based peak search followed by a peak selection based on average peak height, using multi-reference alignment (MRA). Its extension, using multi-reference multiple alignment (MRMA), was found to enable particle pickup at higher accuracy even from extremely noisy images with a signal-to-noise ratio of 0.001. We refer to these new methods as stochastic pickup with MRA (MRA-StoPICK) or with MRMA (MRMA-StoPICK). MRMA-StoPICK has a higher pickup accuracy and furthermore, is almost independent of parameter settings. They were successfully applied to cryo-electron micrographs of Rice dwarf virus. Because current computational resources and parallel data processing environments allow somewhat CPU-intensive MRA-StoPICK and MRMA-StoPICK to be performed in a short period, these methods are expected to allow high-resolution analysis of the 3D structure of particles.
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    Immunohistochemistry and ultrastructural characteristics of nerve endings in the oral mucosa of rat (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: The sensory nerve endings of the rat tongue, cheek and palate were studied using immunohistochemical staining and transmission electron microscopy analysis. The specimens were fixed in modified Karnovsky solution and embedded in Spurr resin. Substance P, calcitonin gene-related peptide (CGRP)- and protein gene product 9.5 (PGP b9.5)-containing nerve fibers in the rat tongue, cheek and palate were examined by electronic microscopical analysis and immunohistochemical localization. These fibers run very close to the basal lamina of the epithelium and extend into the filliform and fungiform papillae. Numerous plexiform fibers immunoreactive for substance P, CGRP and PGP 9.5 were found in the connective tissue of mucosa. Electron microscopic observations showed clearly immunostained nerve fibers, which are located very close to the basal lamina of epithelial cells. Some electron-dense granules may be observed in the axoplasms of both substance P and CGRP immunoreactive fibers. Several lamellar corpuscles into the subepithelial connective tissue papillae, Merkel corpuscles and numerous thin unmyelinated and myelinated axons were observed. The terminal axons revealed numerous mitochondria, neurofilaments, microtubules and clear vesicles in the base of axoplasmic protrusions. The lamellar cells showed caveolae and interlamelar spaces filled by amorphous substance. Between the lamellar cells and axoplasmic membrane, and in the adjacent lamellae region, desmosome-type junctions were observed. The quantitative and morphometric analysis showed nerve endings with an average area of 4.83 ± 3.4 μm 2 and 19.4 internal mitochondria in this site and the organized corpuscles with an average area of 79.24 ± 27.24 μm 2 and 24.23 internal mitochondria in this place. All the structures observed are involved in the transmission of pain and mechanoreceptors stimulus of these oral mucosae.
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    A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as ‘funicular cabin’ for the cell nucleus, and actin cables as intracellular ‘funicular’ suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.
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    Serial block-face scanning electron microscopy combined with double-axis electron beam tomography provides new insight into cellular relationships (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: To evaluate the advantages of combination of two advanced electron microscopic technologies such as serial block-face scanning electron microscopy and double-axis electron beam tomography, we analyzed the three-dimensional morphology of cellular relationships between dendritic and plasma cells in the synovial membrane from patients with rheumatoid arthritis, using the combined approach.
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    Ultrathin specimen preparation by a low-energy Ar-ion milling method (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: The low-energy Ar-ion milling method was used to prepare ultrathin specimens for transmission electron microscope observation. The samples were thinned initially by a usual focused ion beam technique or typical Ar-ion milling with a high energy of 2–10 keV and were thinned additionally by an Ar-ion beam with an energy less than 1 keV, typically 500–900 eV. This low-energy ion beam was scanned over the specimen, and secondary electrons induced by the ion beam could be detected to form secondary electron images with a resolution of a few micrometre. Because a desired area can be selected and thinned by the low-energy ion beam, redeposition or cross contamination from irradiation of a metal grid that supports the sample can be prevented. It was shown that the low-energy Ar-ion beam thins a surface amorphous damage layer preferentially and effectively rather than a crystal specimen. Images from ultrathin specimens of two different materials revealed a detailed structure.
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    Subscription Page (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
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    Cover Page (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
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    Restoration of singularities in reconstructed phase of crystal image in electron holography (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: Off-axis electron holography can be used to measure the inner potential of a specimen from its reconstructed phase image and is thus a powerful technique for materials scientists. However, abrupt reversals of contrast from white to black may sometimes occur in a digitally reconstructed phase image, which results in inaccurate information. Such phase distortion is mainly due to the digital reconstruction process and weak electron wave amplitude in some areas of the specimen. Therefore, digital image processing can be applied to the reconstruction and restoration of phase images. In this paper, fringe reconnection processing is applied to phase image restoration of a crystal structure image. The disconnection and wrong connection of interference fringes in the hologram that directly cause a 2 phase jump imperfection are correctly reconnected. Experimental results show that the phase distortion is significantly reduced after the processing. The quality of the reconstructed phase image was improved by the removal of imperfections in the final phase.
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    Response of thymus lymphocytes to streptozotocin-induced diabetes and exogenous vitamin C administration in rats (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: Diabetes causes oxidative stress, which in turn generates excessive free radicals resulting in cellular damage. Vitamin C is an antioxidant that protects tissues and organs from oxidative stress. The thymus is one of the most important lymphoid organs, which regulates T-lymphocyte proliferation and maturation. The aim of this study is to investigate the protective effects of vitamin C on the thymus of streptozotocin (STZ)-induced diabetic rats. The mitotic activity and cell integrity of thymic lymphocytes were explored. Wistar Albino rats were divided into three groups: control (Group 1), STZ-diabetes (Group 2) and vitamin C-treated STZ-diabetics (Group 3). Rats received a single intraperitoneal injection of 45 mg/kg STZ to induce diabetes. Vitamin C (20 mg/kg) was administered intragastrically. Semithin and ultrathin sections were examined under a light or an electron microscope, respectively. Considerable numbers of mitotic lymphocytes were observed in the thymus of control rats. In the diabetic rats, however, numbers of mitotic lymphocytes decreased to ~57% of controls, and cell division abnormalities were observed. Additionally, diabetic rats showed degeneration in the structure of the thymus including trabecular thickening, accumulation of lipid vacuoles, heterochromatic nuclei and loss of mitochondrial cristae. Degradation of medullar and cortical integrity was also detected. In the vitamin C-treated STZ-diabetic group, the structure of the thymus and mitotic activity of the lymphocytes were similar to the control group. These results suggest that vitamin C protects the thymus against injury caused by diabetes and restores thymocyte mitotic activity.
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    Subscription Page (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
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    Stability of {111}Pd/{0002}ZnO polar interface formed by internal oxidation of Pd-Zn alloys (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: We investigated the stability of Pd/ZnO polar interfaces formed by internal oxidation of Pd–Zn alloys by using high-resolution transmission electron microscopy, electron energy-loss spectroscopy and convergent-beam electron diffraction. At 1273 K, a $$(111{)}_{\mathrm{Pd}}/(000\overline{2}{)}_{\mathrm{ZnO}}$$ polar interface defaceted and transformed into a curved interface, while another (111) Pd /(0002) ZnO polar interface retained its flatness. The $$(111{)}_{\mathrm{Pd}}/(000\overline{2}{)}_{\mathrm{ZnO}}$$ polar interface lost some stability over non-polar interfaces at 1273 K, while the (111) Pd /(0002) ZnO polar interface remained stable.
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    Interaction of electrons with light metal hydrides in the transmission electron microscope (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: Transmission electron microscope (TEM) observation of light metal hydrides is complicated by the instability of these materials under electron irradiation. In this study, the electron kinetic energy dependences of the interactions of incident electrons with lithium, sodium and magnesium hydrides, as well as the constituting element effect on the interactions, were theoretically discussed, and electron irradiation damage to these hydrides was examined using in situ TEM. The results indicate that high incident electron kinetic energy helps alleviate the irradiation damage resulting from inelastic or elastic scattering of the incident electrons in the TEM. Therefore, observations and characterizations of these materials would benefit from increased, instead decreased, TEM operating voltage.
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    The presence of perforated synapses in the striatum after dopamine depletion, is this a sign of maladaptive brain plasticity? (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: Synaptic plasticity is the process by which long-lasting changes take place at synaptic connections. The phenomenon itself is complex and can involve many levels of organization. Some authors separate forms into adaptations that have positive or negative consequences for the individual. It has been hypothesized that an increase in the number of synapses may represent a structural basis for the enduring expression of synaptic plasticity during some events that involve memory and learning; also, it has been suggested that perforated synapses increase in number after some diseases and experimental situations. The aim of this study was to analyze whether dopamine depletion induces changes in the synaptology of the corpus striatum of rats after the unilateral injection of 6-OHDA. The findings suggest that after the lesion, both contralateral and ipsilateral striata exhibit an increased length of the synaptic ending in ipsilateral (since third day) and contralateral striatum (since Day 20), loss of axospinous synapses in ipsilateral striatum and a significant increment in the number of perforated synapses, suggesting brain plasticity that might be deleterious for the spines, because this type of synaptic contacts are presumably excitatory, and in the absence of the modulatory effects of dopamine, the neuron could die through excitotoxic mechanisms. Thus, we can conclude that the presence of perforated synapses after striatal dopamine depletion might be a form of maladaptive synaptic plasticity.
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    Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein–protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques.
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    Cover Page (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
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    Atomic force microscope-assisted scanning tunneling spectroscopy under ambient conditions (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: We have developed a method of atomic force microscopy (AFM)-assisted scanning tunneling spectroscopy (STS) under ambient conditions. An AFM function is used for rapid access to a selected position prior to performing STS. The AFM feedback is further used to suppress vertical thermal drift of the tip–sample distance during spectroscopy, enabling flexible and stable spectroscopy measurements at room temperature.
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    In situ transmission electron microscopy observation of dislocation motion in 9Cr steel at elevated temperatures: influence of shear stress on dislocation behavior (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: To elucidate high-temperature plastic deformation (creep) mechanism in materials, it is essential to observe dislocation motion under tensile loading. There are many reports on in situ transmission electron microscopy (TEM) observations in the literature; however, the relationship between the dislocation motion and shear stress in 9Cr steel is still not clear. In this study, in order to evaluate this relationship quantitatively, in situ TEM observations were carried out in conjunction with finite element method (FEM) analysis. A tensile test sample was strained at an elevated temperature (903 K) inside a transmission electron microscope, and the stress distribution in the strained sample was analyzed by FEM. The dislocation behavior was clearly found to depend on the shear stress. At a shear stress of 66 MPa, both the dislocation velocity and mobile dislocation density were low. However, a high shear stress level of 95 MPa caused a noticeable increase in the dislocation velocity and mobile dislocation density. Furthermore, in this article, we discuss the dependence of the dislocation behavior on stress. The results presented here also indicate that the relationship between the microstructure and the strength of materials can be revealed by the methods used in this work.
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    Dual-axis 360{degrees} rotation specimen holder for analysis of three-dimensional magnetic structures (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: A dual-axis 360° rotation specimen holder was developed for use in reconstructing the three-dimensional (3D) distribution of a magnetic field using a combination of electron holography and tomography. Pillar-shaped specimens are used to obtain accurate reconstruction without a missing angle. The holder's rotation rod can be turned 〉360°; the pillar is set ±45° to the azimuth for both x - and y -axis rotation. Two rotation series of holograms in individual axes are recorded for vector field tomography. The two vector components of the magnetic field are reconstructed directly from the two series of holograms, and the remaining component is calculated using Maxwell's equation, div B = 0. As a result, all 3D magnetic fields are reconstructed.
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    Development of an electrochemical cell for in situ transmission electron microscopy observation (2014)
    Oxford University Press
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    Publication Date: 2014-11-29
    Description: A new type of electrochemical cell was developed for in situ transmission electron microscopy observation that enables the electrode materials to be conveniently changed. The electrochemical cell was used to observe the electrochemical growth or dissolution of copper islands on a gold film with simultaneous cyclic voltammetry measurements. The copper islands could be explained by three-dimensional growing model.
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    The vasohibin family: a novel family for angiogenesis regulation (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Angiogenesis, a formation of neovessels, is regulated by the local balance between angiogenesis stimulators and inhibitors. A number of such endogenous regulators of angiogenesis have been found in the body. Recently, vasohibin-1 (VASH1) was isolated as a negative feedback regulator of angiogenesis produced by endothelial cells (ECs) and subsequently vasohibin-2 (VASH2) as a homologue of VASH1. It was then explored that VASH1 is expressed in ECs to terminate angiogenesis, whereas VASH2 is expressed in cells other than ECs to promote angiogenesis in the mouse model of angiogenesis. This review will focus on the vasohibin family members, which are novel regulators of angiogenesis.
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    Topics: Biology , Chemistry and Pharmacology
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    Activation of the Wnt/{beta}-catenin pathway and tissue inhibitor of metalloprotease 1 during tertiary dentinogenesis (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Tertiary dentin is deposited inside teeth after various stimuli and serves as a major defensive wall to preserve pulp cells. However, the molecular mechanisms of the activation of quiescent odontoblasts, immature pulp cells and tertiary dentin formation are still unclear. Therefore, we performed a comprehensive gene expression analysis of pulp cells after cavity preparation of 9-week-old rat molars to clarify the critical molecules in tertiary dentinogenesis. As a result, mRNA expression of various molecules was up- or down-regulated. Notably, several members of the matrix metalloprotease family and their endogenous inhibitors were up-regulated after cavity preparation. In situ hybridization showed that tissue inhibitor of metalloprotease 1 ( Timp1 ) was widely and continuously distributed in the pulp beneath the cavity in vivo . We also observed accumulation of β-catenin in the pulp cells beneath the cavity by fluorescence immunohistochemistry. Furthermore, Timp1 transcription was repressed by a dominant-negative TCF4 in immature undifferentiated mesenchymal cells, but not altered in mature odontoblast-like cells. These results indicate that cavity preparation may activate the Wnt/β-catenin pathway and the Wnt/β-catenin pathway and Timp1 may be correlatively involved in pulp repair. Timp1 might play crucial roles in reactivation of immature pulp cells for tertiary dentinogenesis.
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    Phosphoinositide 5-phosphatases: how do they affect tumourigenesis? (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: The activity of biological molecules is often affected by their phosphorylation state. Regulatory phosphorylation operates as a binary switch and is usually controlled by counteracting kinases and phosphatases. However, phosphatidylinositol (PtdIns) has three phosphorylation sites on its inositol ring. The phosphorylation status of PtdIns is controlled by multiple kinases and phosphatases with distinct substrate specificities, serving as a ‘lipid code’ or ‘phosphoinositide code’. Class I phosphoinositide 3-kinase (PI3K) converts PtdIns(4,5)P 2 to PtdIns(3,4,5)P 3 , which plays a pivotal role in signals controlling glucose uptake, cytoskeletal reorganization, cell proliferation and apoptosis. PI3K is pro-oncogenic, whereas phosphoinositide phosphatases that degrade PtdIns(3,4,5)P 3 are not always anti-oncogenic. Recent studies have revealed the unique characteristics of phosphoinositide 5-phosphatases.
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    Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Vascular endothelial growth factors (VEGFs) belong to the platelet-derived growth factor supergene family, and they play central roles in the regulation of angiogenesis and lymphangiogenesis. VEGF-A, the major factor for angiogenesis, binds to two tyrosine kinase (TK) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and regulates endothelial cell proliferation, migration, vascular permeability, secretion and other endothelial functions. VEGFR-2 exhibits a strong TK activity towards pro-angiogenic signals, whereas the soluble VEGFR-1 (sFlt-1) functions as an endogenous VEGF inhibitor. sFlt-1 is abnormally overexpressed in the placenta of preeclampsia patients, resulting in the major symptoms of the disease due to abnormal trapping of VEGFs. The VEGF-VEGFR system is crucial for tumour angiogenesis, and anti-VEGF-VEGFR molecules are now widely used in the clinical field to treat cancer patients. The efficacy of these molecules in prolonging the overall survival of patients has been established; however, some cancers do not respond well and reduced tumour sensitivity to anti-VEGF signals may occur after long-term treatment. The molecular basis of tumour refractoriness should be determined to improve anti-angiogenic therapy.
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    Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatography–electrospray ionization time-of-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.
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    Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination (2012)
    Oxford University Press
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    Publication Date: 2012-12-22
    Description: Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.
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    Single-molecule imaging with a tagged ribosome to explore trans-translation (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Single-molecule imaging is a powerful technique to visualize molecular interactions and movements. Translation is one of the most interesting targets for researchers with the molecular-imaging skills, since mRNA, tRNA and translation factors interact with or move inside or on the ribosome in an ordered manner. Trans -translation is a bacterial quality control system to rescue the ribosomes stalled at the 3' end of the mRNA, and this phenomenon is recapitulated in vitro with defined factors including two trans -translation-specific entities tmRNA and SmpB. Zhou et al. (Single molecule imaging of the trans -translation entry process via anchoring of the tagged ribosome. J Biochem 2011;149:609-618.) successfully visualized the interaction of the tmRNA–SmpB complex with the ribosome by immobilizing the ribosome on the quartz surface with the HaloTag technology. This ribosome-anchoring system may be useful for the imaging analysis of other processes of translation.
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    The functional roles of S1P in immunity (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: The lipid mediator sphingosine-1-phosphate (S1P) is generated within cells from sphingosine by two sphingosine kinases (SPHK1 and SPHK2). Intracellularly synthesized S1P is released into the extracellular fluid by S1P transporters, including SPNS2. Released S1P binds specifically to the G protein-coupled S1P receptors (S1PR1/S1P 1 –S1PR5/S1P 5 ), which activate a diverse range of downstream signalling pathways. Recent studies have proposed that one of the central physiological functions of intercellular S1P signalling is in lymphocyte trafficking in vivo because genetic disruption of SPHK1/2, SPNS2 or S1PR1/S1P 1 in mice induces a lymphopenia phenotype. In this review, we discuss the current understanding of intercellular S1P signalling in the context of immunity.
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    Cdc6: a trifunctional AAA+ ATPase that plays a central role in controlling the G1-S transition and cell survival (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Cdc6 is the AAA+ ATPase that assembles prereplicative complexes on replication origins in eukaryotic chromosomes. Recently, the same Cdc6 protein was found to exert two more functions in mammalian cells to promote cell proliferation and survival: ATP-dependent activation of p21 CIP1 - or p27 KIP1 -bound Cdk2-cyclin A/E complexes and obstruction of apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules. These findings not only redefined the biological role of mammalian Cdc6 but also led the discovery of entirely new mechanisms controlling Cdk2 activity and apoptosis. This review focuses on this multi-functional AAA+ ATPase and the newly discovered mechanisms by which it controls the G 1 –S transition and cell survival during proliferation.
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    Pseudomonas putida PydR, a RutR-like transcriptional regulator, represses the dihydropyrimidine dehydrogenase gene in the pyrimidine reductive catabolic pathway (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: The pyrimidine reductive catabolic pathway is important for the utilization of uracil and thymine as sources of nitrogen and carbon. The pathway is controlled by three enzymes: dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase and β-alanine synthase. The putative DPD genes, pydX and pydA , are tandemly arranged in the Pseudomonas putida genome. Intriguingly, a putative transcriptional regulator, PydR, homologous to Escherichia coli RutR, a repressor of the Rut-dependent pyrimidine degradation pathway, is located downstream of pydX and pydA . In this study, we show that a pydA strain of P. putida fails to grow on a minimal media containing uracil or thymine as a sole nitrogen source, demonstrating the physiological importance of DPD in the reductive pathway. The expression of pydA and DPD activity in the absence of uracil were significantly higher in a pydR strain than in the wild-type strain, indicating that PydR acts as a repressor of the pyrimidine reductive pathway in P. putida . Phylogenetic analysis of RutR and PydR suggests that these homologous repressors may have evolved from a common ancestral protein that regulates pyrimidine degradation.
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    Mediator lipidomics in acute inflammation and resolution (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Acute inflammation is an indispensable host response to foreign challenges or tissue injury. In healthy conditions, inflammatory processes are self-limiting and self-resolving, suggesting the existence of endogenous mechanisms for the control of inflammation and resolution. A comprehensive understanding of the cellular and molecular events of a well-orchestrated inflammatory response is required, and recent studies have uncovered the roles of endogenous lipid mediators derived from polyunsaturated fatty acids (i.e. lipoxins, resolvins, protectins) in controlling the resolution of inflammation. This review presents recent advances in understanding the formation and action of these mediators, especially focusing on the LC-MS/MS-based lipidomics approach and the emerging roles of eosinophils and eosinophil-derived lipid mediators in controlling acute inflammation and resolution.
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    Matrix control of transforming growth factor-{beta} function (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: The cytokine transforming growth factor-beta (TGF-β) has multiple effects in both physiological and pathological conditions. TGF-β is secreted as part of a tripartite complex from which it must be released in order to bind to its receptor. Sequestration of latent TGF-β in the extracellular matrix (ECM) is crucial for proper mobilization of the latent cytokine and its activation. However, contrary to expectation, loss-of-function mutations in genes encoding certain matrix proteins that bind TGF-β yield elevated, rather than decreased, TGF-β levels, posing a ‘TGF-β paradox.’ In this review, we discuss recent findings concerning the relationship of TGF-β, ECM molecules, and latent TGF-β activation and propose a model to resolve the ‘TGF-β paradox.’
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    Identification of the catalytic residues of sequence-specific and histidine-free ribonuclease colicin E5 (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Colicin E5 cleaves tRNAs for Tyr, His, Asn and Asp in their anticodons to abolish protein synthesis in Escherichia coli . We previously showed how its C-terminal RNase domain, E5-CRD, recognizes the anticodon bases but the catalytic mechanism remained to be elucidated. Although the reaction products with 5'-OH and 2',3'-cyclic phosphate ends suggested a similar mechanism to those of RNases A and T1, E5-CRD does not have the His residues necessary as a catalyst in usual RNases. To identify residues important for the catalytic reaction, mutants as to all residues within 5 Å from the central phosphorus of the scissile phosphodiester bond were prepared. Evaluation of the killing activities of the mutant colicins and the RNase activities of the mutant E5-CRDs suggested direct involvement of Arg33, Lys25, Gln29 and Lys60 in the reaction. Particularly, Arg33 plays a critical role and Ile94 provides a structural support of Arg33. Crystal structure of the complex of E5-CRD(R33Q)/dGpdUp showed structural and binding functional integrity of this mutant protein, suggesting involvement of Arg33 in the catalytic reaction. The structure of the free E5–CRD, we also determined, showed great flexibility of a flap region, which facilitates the access of Lys60 to the substrate in an induced-fit manner.
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    Sulfatide negatively regulates the fusion process of human parainfluenza virus type 3 (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Sulfatide (HSO 3 -3-galactosylceramide), which enriched in lipid rafts of plasma membranes in various epithelial cell lines, is a critical component of host cells for effective production of influenza A virus. However, the function of sulfatide in other virus infections targeting epithelial cells remains unknown. In this study, the effect of sulfatide on infection of human parainfluenza virus type 3 (hPIV3) was demonstrated by using genetically produced sulfatide-enriched cells and by treatment of hPIV3-infected cells with anti-sulfatide monoclonal antibody (GS-5) as well as by addition of sulfatide to the cells. hPIV3 was found to bind to sulfatide in a virus overlay assay and a solid-phase binding assay. Genetic expression of sulfatide in COS-7 cells defective in sulfatide suppressed initial hPIV3 infection and formation of multinucleate virus-infected cells. Treatment of virus-infected LLC-MK2 cells with GS-5 promoted formation of multinucleate cells. In contrast, exogenous addition of sulfatide to hPIV3-infected COS-7 cells and cells expressing the hPIV3- hemagglutinin-neuraminidase ( HN ) gene and fusion ( F ) gene conspicuously reduced the formation of multinucleate cells. The results suggest that sulfatide negatively regulates the fusion process of hPIV3, possibly through interaction with HN or F glycoprotein on the cell surface.
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    Annexin A3 as a negative regulator of adipocyte differentiation (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Annexin A3 is a protein belonging to the annexin family, and it is mainly present in cellular membranes as a phospholipid-binding protein that binds via the calcium ion. However, its physiological function remains to be clarified. We examined the expression of annexin A3 in mouse tissues and found for the first time that annexin A3 mRNA and its protein were expressed more strongly in adipose tissues than in other tissues. In adipose tissues, annexin A3-expressing cells were present in the stromal vascular fraction, and precisely identical to Pref-1-positive preadipocytes, Pref-1 being an epidermal growth factor repeat-containing transmembrane protein that inhibits adipogenesis. In 3T3-L1 cells, used as a model of adipogenesis, annexin A3 was down-regulated at an early phase of adipocyte differentiation, and this pattern paralleled that of Pref-1. Suppression of annexin A3 in these cells with siRNA caused elevation of the PPAR2 mRNA level and lipid droplet accumulation. In conclusion, our data suggest that annexin A3 is a negative regulator of adipocyte differentiation.
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    PKL01, an Ndr kinase homologue in plant, shows tyrosine kinase activity (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Protein phosphorylation by protein tyrosine (Tyr) kinases plays important roles in a variety of signalling pathways in cell growth, differentiation and oncogenesis in animals. Despite the absence of classical Tyr kinases in plants, a similar ratio of phosphotyrosine residues in phosphorylated proteins was found in Arabidopsis thaliana as in human. However, protein kinases responsible for tyrosine phosphorylation in plants except some dedicated dual-specificity kinases still remain unclear. In this study, we found that PKL01, a nuclear Dbf2-related (Ndr) kinase homologue in Lotus japonicus , was autophosphorylated at a tyrosine residue when it was expressed in Escherichia coli , but kinase-dead mutant of PKL01 was not. Tyrosine phophorylation site in PKL01 was identified as Tyr-56 by LC-MS/MS analysis. Recombinant PKL01, which had been dephosphorylated by an alkaline phosphatase, could be phosphorylated again at the Tyr residue when it was incubated with ATP. Furthermore, other Ndr kinases in plants and PKL01 phosphorylated on Tyr residues in the exogenous substrates such as poly(Glu, Tyr) 4:1 and casein. Therefore, the Ndr kinases in plants, which had been assumed as protein serine (Ser)/threonine (Thr) kinases, turned out to be dual-specificity kinases responsible for phosphorylation of Tyr residues and Ser/Thr residues in plant proteins.
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    The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation (2012)
    Oxford University Press
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    Publication Date: 2012-09-29
    Description: Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols ( trans - and cis -resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans - and cis -resveratrol 3- O -glucuronidation. Although the K m value for trans -resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K m values for cis -resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K m in trans -resveratrol, and V max and K si in cis -form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.
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    JEM in this issue, 61:5 (2012)
    Oxford University Press
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    Publication Date: 2012-10-12
    Print ISSN: 0022-0744
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    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
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    Cover Page (2012)
    Oxford University Press
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    Publication Date: 2012-10-12
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    The role of collagen arrangement change during tendon healing demonstrated by scanning electron microscopy (2012)
    Oxford University Press
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    Publication Date: 2012-10-12
    Description: The dry weight of tendon tissue is accounted for mainly by collagen fibers. Accordingly, the tendon-healing process primarily involves repair of collagen fibers. During the remodeling phase of tendon healing, newly proliferating collagen fibers are transformed into a mature repaired tendon. Despite the importance of this phenomenon, the details of fibrous rebuilding have not been reported previously. The aim of this study was to visualize the ultrastructural changes and to obtain a clear understanding of the reorganization of the collagen fibers in the tendon repair site, using rat Achilles tendons. We used scanning electron microscopy (SEM) with cell maceration as the main method of analysis. Pretreatment with cell maceration removed the cellular components successfully. This allowed precise visualization of each collagen fiber and the three-dimensional network of the fibers. This study was the first to apply the cell-maceration/SEM method to observe tendon tissue. Seven days after surgery, new collagen fibers grew extensively in the repair site in a random arrangement. Fourteen days after surgery, the collagen fibers began to form an axial arrangement. Near the tendon stump, this change progressed from the outer layer to the core region. On the other hand, in the middle of the repair site, it progressed from the core to the outer layer. Change in the axial arrangement of collagen fibers contributes to the connection between the repair site and the tendon stump and to the separation of the repair site from the paratenon.
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    Subscription Page (2012)
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    Mapping of minority carrier lifetime distributions in multicrystalline silicon using transient electron-beam-induced current (2012)
    Oxford University Press
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    Publication Date: 2012-10-12
    Description: We have used transient electron-beam-induced current (EBIC) to map minority carrier lifetime distributions in multicrystalline Silicon (mc-Si). In this technique, the electron beam from a scanning transmission electron microscope was on–off modulated while the sample was scanned. The resulting transient EBIC was analyzed to form a lifetime map. An analytical function was introduced as part of the analysis in determining this map. We have verified this approach using numerical simulations and have reproduced a lifetime map for an mc-Si wafer.
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    Direct observation of the initial process of Ostwald ripening using spherical aberration-corrected transmission electron microscopy (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: We study in situ behavior of platinum single atoms on amorphous carbon (a-carbon) using a spherical aberration-corrected transmission electron microscope (AC-TEM). Diffusion of single atoms, bi-atoms, clusters (〈1 nm) and nanoparticles (〈3 nm) was recorded in the same image with a time resolution of 1 s, and such diffusion matches the expected mechanism of Ostwald ripening, which was seen on these samples. In situ AC-TEM shows promise for dynamical observation of single atom diffusion, which is important for understanding nanosized catalysts and ceramic sintering processes. We apply in situ AC-TEM to image platinum (Pt) nanoparticles on a-carbon, which is a model catalyst system for the real Pt electrode catalysts using alloys and core–shell structures supported on carbon/oxide composite materials in the proton exchange membrane fuel cell.
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    Ultrastructural interaction between spermatozoon and human oviductal cells in vitro (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: The oviduct is an important organ for successful mammalian reproduction. In this work, human oviducts were inseminated and their explants analyzed using scanning electron microscopy in order to study, at a finer ultrastructual level, the interaction between spermatozoon and oviduct in vitro . Results show unequivocally a spermatozoon tightly attached through the acrosomal region of its head to several cilia of the human tubal epithelial cells. This finding proves that spermatozoa do indeed adhere to the endosalpinx, a fact of utmost relevance for the physiology of the reproductive process, since it supports the idea of a spermatozoa reservoir being formed in the oviduct, which is also briefly discussed.
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    Efficient and accurate analysis of mitochondrial morphology in a whole cell with a high-voltage electron microscopy (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: Mitochondria in all eukaryotes are essential organelles responsible for adenosine triphosphate synthesis, calcium homeostasis and steroidogenesis. Because the structure and distribution of mitochondria are highly diverse depending on their function and cellular conditions, it is important to develop a rapid and accurate method to assess their morphology. In this study, we visualize whole mitochondria in cultured cells using high-voltage electron microscopy (HVEM). Compared with conventional transmission electron microscopic approaches, the present method does not require thin sectioning and thus requires less time for image acquisition and processing. Furthermore, compared with fluorescence-based light microscopic approaches, our method provides more accurate size information. Thus, we propose that HVEM is a useful tool for rapid and accurate analysis of mitochondrial morphology and distribution in a cell.
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    A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.
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    Subscription Page (2012)
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    Publication Date: 2012-04-16
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    Magnetization distribution of magnetic vortex of amorphous FeSiB investigated by electron holography and computer simulation (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: The three-dimensional spin structure of the magnetic vortex of FeSiB, an amorphous soft magnetic material, was investigated by holography observation and computer simulation. Magnetization distribution in the neighborhood of the vortex center was estimated from the phase distribution obtained by holography observation. To confirm this magnetization distribution, sample-tilting experiments were performed: when the sample was tilted with respect to the electron beam direction, the phase-image center was found to shift along the tilting axis. Finite-element computer simulation was carried out to estimate the amount of shifts of the phase-image center in the sample tilting from the experimental magnetization distributions in the no sample-tilting conditions. We found that the simulated shifts of the phase-image center were in good agreement with those in the sample-tilting experiment, thus confirming the magnetization distribution near the vortex center obtained by holography observation.
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    JEM in this issue, 61:2 (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
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    Cover Page (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
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    Space charge characteristics of an insulating thin film negatively charged by a low-energy electron beam (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: In this study, based on a comprehensive numerical simulation of self-consistent charging, we investigate the formation, evolution and influencing factors of space charge distributions for a grounded insulating thin film of SiO 2 negatively charged by a keV non-penetrating focused electron beam. The simulated space charge presents first positive distributions and then negative ones along both the radial and depth directions because of the difference between electron and hole transports. The variations in distribution occur within a range of the minimum potential acting as a potential barrier for carrier transport. The negative space charge is distributed more widely and deeply, though its peak value in density is usually lower than that of the positive one. Electrons trapped outside the minimum potential range dominate the strength of negative charging. With the increase in potential barrier and the occurrence of leakage current, the space charge eventually reaches equilibrium and exhibits an approximately one-dimensional axial distribution outside the minimum potential range. Distribution features of the space charge density in the equilibrium state correlate with the film and beam parameters via transients of the leakage current. These results and analyses provide new insights into the negative charging effects involved in various electron-beam-based surface microscopic methods, analyses and fabrication techniques.
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    Structure and formation of the twisted plywood pattern of collagen fibrils in rat lamellar bone (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: This study was designed to elucidate details of the structure and formation process of the alternate lamellar pattern known to exist in lamellar bone. For this purpose, we examined basic internal lamellae in femurs of young rats by transmission and scanning electron microscopy, the latter employing two different macerations with NaOH at concentrations of 10 and 24%. Observations after the maceration with 10% NaOH showed that the regular and periodic rotation of collagen fibrils caused an alternation between two types of lamellae: one consisting of transversely and nearly transversely cut fibrils, and the other consisting of longitudinally and nearly longitudinally cut fibrils. This finding confirms the consistency of the twisted plywood model. The maceration method with 24% NaOH removed bone components other than cells, thus allowing for three-dimensional observations of osteoblast morphology. Osteoblasts extended finger-like processes paralleling the inner bone surface, and grouped in such a way that, within a group, the processes arranged in a similar direction. Transmission electron microscopy showed that newly deposited fibrils were arranged alongside these processes. For the formation of the alternating pattern, our findings suggest that: (1) osteoblasts control the collagen fibril arrangement through their finger-like process position; (2) osteoblasts behave similarly within a group; (3) osteoblasts move their processes synchronously and periodically to promote alternating different fibril orientation; and (4) this dynamic sequential deposition of fibrils results in the alternate lamellar (or twisted plywood) pattern.
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    Three-dimensional reconstructions of electrostatic potential distributions with 1.5-nm resolution using off-axis electron holography (2012)
    Oxford University Press
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    Publication Date: 2012-04-16
    Description: Three-dimensional (3D) reconstruction experiments were carried out by observing high-resolution 3D electrostatic potential distributions of Pt nanoparticles using off-axis electron holographic tomography. These Pt nanoparticles were mounted on the surfaces of amorphous silicon pillars. In order to realize high-resolution observation, we developed a mechanically stable 3D specimen holder with small specimen drifts and vibrations. From the 3D electrostatic potential distribution data of Pt nanoparticles (2.0 nm in diameter), we obtained the resolution of 1.5 nm.
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    Role of PKC-{alpha} in NF-{kappa}B-MT1-MMP-mediated activation of proMMP-2 by TNF-{alpha} in pulmonary artery smooth muscle cells (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We sought to evaluate the mechanism(s) associated with pro matrix metalloprotease 2 (proMMP-2) activation in bovine pulmonary artery smooth muscle cells. Preincubation of cells with anti-TNFR1 antibody prevented tumour necrosis factor-α (TNF-α)-induced proMMP-2 activation and increase in membrane type 1 matrix metalloprotease (MT1-MMP) expression as well as inhibition of tissue inhibitor of metalloproteinase 2 (TIMP-2) expression, indicating the role of TNFR1 receptor during TNF-α stimulation. Anti-MT1-MMP antibody abrogated proMMP-2 activation by TNF-α-stimulated cell membrane, suggesting the involvement of MT1-MMP in proMMP-2 activation. Induction of MT1-MMP expression in response to TNF-α occurs via activation of nuclear factor (NF)-B on inhibitory B kinase (IKK) activation and subsequently phosphorylation/degradation of IB-α. Inhibition of protein kinase C (PKC)-α activity by Go6976 and PKC-α siRNA prevented TNF-α-induced IKK activity, IB-α phosphorylation/degradation and NF-B activation. Inhibition of PKC-α activity also prevented TNF-α-induced MT1-MMP expression and proMMP-2 activation as well as down regulation of TIMP-2 expression. Inhibition of IB-α phosphorylation by PS-1145, an IKK selective inhibitor, prevented TNF-α-induced increase in MT1-MMP expression and proMMP-2 activation, which although did not alter inhibition of TIMP-2 expression. Overall, we unravelled a hitherto unknown mechanism of the involvement of PKC-α in proMMP-2 activation and inhibition of TIMP-2 expression by NF-B–MT1-MMP–dependent and –independent pathway, respectively, during TNF-α stimulation in pulmonary artery smooth muscle cells.
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    Tumour endothelial cells acquire drug resistance in a tumour microenvironment (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: Tumour growth is dependent on angiogenesis, and tumour blood vessels are recognized as an important target for cancer therapy. Tumour endothelial cells (TECs) are the main targets of anti-angiogenic therapy. Unlike the traditionally held view, some TECs may be genetically abnormal and might acquire drug resistance. Therefore, we investigated the drug resistance of TECs and the mechanism by which it is acquired. TECs show resistance to paclitaxel through greater mRNA expression of multidrug resistance 1, which encodes P-glycoprotein, as compared with normal endothelial cells. We found that high levels of vascular endothelial growth factor in tumour-conditioned medium may be responsible for upregulated P-glycoprotein expression. This is a novel mechanism for the acquisition of drug resistance by TECs in a tumour microenvironment. This review focuses on the possibility that TECs can acquire drug resistance.
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    Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem . 148, 593–602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis . Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC 50 value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.
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    Transforming growth factor-{beta}1 induces epithelial-mesenchymal transition and integrin {alpha}3{beta}1-mediated cell migration of HSC-4 human squamous cell carcinoma cells through Slug (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We investigated whether transforming growth factor (TGF)-β1 promoted epithelial–mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-β target genes expression were most clearly upregulated following TGF-β1 stimulation in HSC-4 cells, indicating that HSC-4 cells were the most responsive to TGF-β1. In addition, the expression levels of the mesenchymal markers N-cadherin and vimentin were most clearly induced in HSC-4 cells among the hOSCC cell lines by TGF-β1 stimulation. Interestingly, E-cadherin and β-catenin at the cell surface were internalized in HSC-4 cells stimulated with TGF-β1. In addition, the expression levels of the EMT-related transcription factor Slug was significantly upregulated on TGF-β1 stimulation. Moreover, the downregulation of Slug by RNA interference clearly inhibited the TGF-β1-induced expression of mesenchymal marker and the migration of HSC-4 cells. Proteomics analysis also revealed that the expression levels of integrin α3β1-targeted proteins were upregulated in TGF-β1-stimulated HSC-4 cells. Neutral antibodies against integrin α3 and β1, as well as a focal adhesion kinase (FAK) inhibitor, clearly suppressed TGF-β1-induced cell migration. These results suggest that the EMT and integrin α3β1/FAK pathway–mediated migration of TGF-β1-stimulated HSC-4 hOSCC cells is positively controlled by Slug.
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    All members of the EPI64 subfamily of TBC/RabGAPs also have GAP activities towards Ras (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: The importance of interconnective signalling networks between distinct GTPases and their regulators is being recognized. EPI64C/TBC1D10C/carabin, a haematopoietically enriched GTPase-activating protein (GAP) for Rab35, has been shown to exhibit RasGAP activity. Owing to the diverged Rab specificities among the EPI64 members (EPI64A–C) and the relatively weak sequence conservation between EPI64A/B and EPI64C in their catalytic TBC domains, it is difficult to predict whether EPI64A and B will also have RasGAP activities. Therefore, in this study, we examined the RasGAP activities of all three EPI64 subfamily members. We found that EPI64A–C exhibited in vivo GAP activities towards Ras using three independent methods, spectrofluorometry with Förster resonance energy transfer (FRET) sensors, the Bos' pull-down assay and time-lapse FRET imaging. EPI64A and B were predominantly localized at the periphery of COS-7 cells. In COS-7 cells, confocal FRET imaging showed that H-Ras activity was higher at the Golgi than at the plasma membrane. Thus, we propose that EPI64A and B, which are ubiquitously expressed members of the EPI64 subfamily, inactivate Ras and certain Rabs at the periphery of cells.
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    Granting specificity for breast cancer cells using a hepatitis B core particle with a HER2-targeted affibody molecule (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: Capsid-like particles consisting of a hepatitis B core (HBc) protein have been studied for their potential as carriers for drug delivery systems (DDS). The hollow HBc particle, which is formed by the self-assembly of core proteins comprising 183 aa residues, has the ability to bind to various cells non-specifically via the action of an arginine-rich domain. In this study, we developed an engineered HBc particle that specifically recognizes and targets human epidermal growth factor receptor-related 2 (HER2)-expressing breast cancer cells. To despoil the non-specific binding property of an HBc particle, we genetically deleted the C-terminal 150–183 aa part of the core protein that encodes the arginine-rich domain (HBc). Then, we genetically inserted a Z HER2 affibody molecule into the 78–81 aa position of the core protein to confer the ability of target-cell-specific recognition. The constructed Z HER2 -displaying HBc (Z HER2 -HBc) particle specifically recognized HER2-expressing SKBR3 and MCF-7 breast cancer cells. In addition, the Z HER2 -HBc particle exhibited different binding amounts in accordance with the HER2 expression levels of cancer cells. These results show that the display of other types of affibody molecules on HBc particles would be an expandable strategy for targeting several kinds of cancer cells that would help enable a pinpoint DDS.
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    Preparation and characterization of a monoclonal antibody against the refolded and functional extracellular domain of rat P2X4 receptor (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: The P2X4 purinergic receptor is a key molecule in neuropathic pain, particularly in the allodynia after peripheral nerve injury. We therefore sought to establish an anti-P2X4 receptor monoclonal antibody that would be useful for detection and characterization of the P2X4 receptor. We first prepared the refolded extracellular domain of the rat P2X4 receptor expressed in Escherichia coli . Then, we established a B-cell hybridoma producing the monoclonal antibody for the head domain of the rat P2X4 receptor with strict recognition, including S-S bond formation. In addition, we succeeded in the detection and immune precipitation of rat P2X4 receptor molecules on cultured cells. As the antibody specifically binds to the rat P2X4 receptor molecule, it is expected that the established monoclonal antibody will be applicable as a tool for detecting increasing expression levels of the P2X4 receptor molecule accompanied with increasing intensity of neuropathic pain.
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    Fluorescence imaging of synapse formation and remodeling (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: Brain function is based on proper connectivity between neuronal cells. In the developing brain, neurons extend axons and form synaptic connections with appropriate postsynaptic neurons. Molecular mechanisms underlying establishment of proper synaptic connections are one of the most important topics in the field of developmental neurobiology. Dynamics of synaptic structure and local recruitment of synaptic molecules can be studied by live-cell imaging of neurons expressing fluorescent probes of synaptic molecules. In this review, examples of live-cell fluorescence imaging are presented and their contributions to our understanding about the molecular mechanisms of synapse formation and remodeling are discussed. Imaging of synaptic proteins in living neurons revealed rapid formation of individual synapses within hours and extensive remodeling of synaptic connections. Different types of neurons express unique protrusions from dendrites and axons, which play important roles in synapse formation and maturation. Rapid formation of synaptic structure is associated with continual assembly and disassembly of synaptic scaffolding proteins, which are essential building blocks of the presynaptic active zone and the postsynaptic density (PSD). Quantitative analyses of PSD scaffolding proteins further confirmed their essential roles in maintenance of the synaptic structure. These examples clearly indicate that fluorescence-based live-cell imaging is an indispensable technique in the research on synapse development and its impact will further increase in combination with development of new light microscopic techniques in the future.
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    High-speed atomic force microscopy (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: High-speed atomic force microscopy (HS-AFM) has been developed as a nano-dynamics visualization technique. This microscopy permits direct observation of structure dynamics and dynamic processes of biological molecules in physiological solutions, at a subsecond to sub-100 ms temporal resolution and an ~2 nm lateral and a 0.1 nm vertical resolution. Importantly, tip–sample interactions do not disturb the biomolecules' functions. Various functioning proteins including myosin V walking on an actin filament and bacteriorhodopsin responding to light have been successfully visualized with HS-AFM. In the quest for understanding the functional mechanisms of proteins, inferences no longer have to be made from static snapshots of molecular structures and dynamic behavior of optical markers attached to proteins. High-resolution molecular movies obtained from HS-AFM observations reveal the details of molecules' dynamic behavior in action, without the need for intricate analyses and interpretations. In this review, I first describe the fundamentals behind the achieved high imaging rate and low invasiveness to samples, and then highlight recent imaging studies. Finally, future studies are briefly described.
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    Confessions of an icosahedral virus crystallographer (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: This is a personal history of my structural studies of icosahedral viruses that evolved from crystallographic studies, to hybrid methods with electron cryo-microscopy and image reconstruction (cryoEM) and then developed further by incorporating a variety of physical methods to augment the high resolution crystallographic studies. It is not meant to be comprehensive, even for my own work, but hopefully provides some perspective on the growth of our understanding of these remarkable biologic assemblies. The goal is to provide a historical perspective for those new to the field and to emphasize the limitations of any one method, even those that provide atomic resolution information about viruses.
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    Enabling direct nanoscale observations of biological reactions with dynamic TEM (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: Biological processes occur on a wide range of spatial and temporal scales: from femtoseconds to hours and from angstroms to meters. Many new biological insights can be expected from a better understanding of the processes that occur on these very fast and very small scales. In this regard, new instruments that use fast X-ray or electron pulses are expected to reveal novel mechanistic details for macromolecular protein dynamics. To ensure that any observed conformational change is physiologically relevant and not constrained by 3D crystal packing, it would be preferable for experiments to utilize small protein samples such as single particles or 2D crystals that mimic the target protein's native environment. These samples are not typically amenable to X-ray analysis, but transmission electron microscopy has imaged such sample geometries for over 40 years using both direct imaging and diffraction modes. While conventional transmission electron microscopes (TEM) have visualized biological samples with atomic resolution in an arrested or frozen state, the recent development of the dynamic TEM (DTEM) extends electron microscopy into a dynamic regime using pump-probe imaging. A new second-generation DTEM, which is currently being constructed, has the potential to observe live biological processes with unprecedented spatiotemporal resolution by using pulsed electron packets to probe the sample on micro- and nanosecond timescales. This article reviews the experimental parameters necessary for coupling DTEM with in situ liquid microscopy to enable direct imaging of protein conformational dynamics in a fully hydrated environment and visualize reactions propagating in real time.
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    Transmission electron microscope as an ultimate tool for nanomaterial property studies (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: In this review, a non-standard application of high-resolution transmission electron microscope (HRTEM), namely the creation of so-called NanoLaboratory for the nanomaterial property studies within its pole piece, is presented. The most modern research trends with respect to nanotube, graphene and nanowire, as well as electrical, mechanical and electromechanical properties are demonstrated. In addition, the unique possibilities of modeling real technological processes inside HRTEM, for example, the performance of Li-ion batteries, are illustrated. The contribution particularly highlights the recent research endeavors of our Tsukuba group in line with all the above-mentioned directions of in situ TEM.
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    3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of 〉300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.
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    Fluorescence imaging in the last two decades (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: In commemoration of the 20th anniversary of the molecular cloning of the gene for the green fluorescent protein from the jellyfish Aequorea victoria , I would like to reflect on the development of new fluorescence imaging technology in the last two decades. As this technology has become increasingly diversified, it has become more and more of a challenge to come up with a comprehensive and exhaustive review of it. Here I will focus on optogenetics and large-scale, three-dimensional reconstruction. Those two technological innovations have been achieved in the neuroscience community owing to the combined efforts of molecular biologists and light microscopists. In addition, modern fluorescence imaging has indeed improved our understanding of the spatiotemporal regulation of fundamental biological functions at cellular level. As an example, I will introduce some findings we made regarding the movement of biomolecules across the nuclear membrane. The above-mentioned imaging approaches are possible today but were impossible two decades ago.
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    A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: We developed an efficient method for introduction of 3-azidotyrosine (N 3 -Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA ( CUA) , and made an orthogonal tRNA (CUA) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N 3 -Y was selected. We then expressed rat calmodulin (CaM) containing N 3 -Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N 3 -Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N 3 -Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA ( CUA) gene. Although the yields of full-length CaM increased ~ 3-fold, the ratio of N 3 -Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N 3 -Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N 3 -Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.
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    Pharmacophore identification of ascofuranone, potent inhibitor of cyanide-insensitive alternative oxidase of Trypanosoma brucei (2013)
    Oxford University Press
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    Publication Date: 2013-02-13
    Description: Trypanosoma brucei is a parasite that causes human African trypanosomiasis (HAT). The parasites depend on the cyanide-insensitive trypanosome alternative oxidase (TAO) for their vital aerobic respiration. Ascofuranone (AF), a potent and specific sub-nanomolar inhibitor of the TAO quinol oxidase, is a potential novel drug with selectivity for HAT, because mammalian hosts lack the enzyme. To elucidate not only the inhibition mechanism but also the inhibitor–enzyme interaction, AF derivatives were designed and synthesized, and the structure–activity relationship was evaluated. Here we identified the pharmacophore of AF that interacts with TAO. The detailed inhibitory profiles indicated that the 1-formyl and 6-hydroxyl groups, which might contribute to intramolecular hydrogen bonding and/or serve as hydrogen-bonding donors, were responsible for direct interaction with the enzyme.
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    Microscopy in this issue (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
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    Preface on the occasion of the inauguration issue of Microscopy (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 94
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    Monochromated STEM with a 30 meV-wide, atom-sized electron probe (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: The origins and the recent accomplishments of aberration correction in scanning transmission electron microscopy (STEM) are reviewed. It is remembered that the successful correction of imaging aberrations of round lenses owes much to the successful correction of spectrum aberrations achieved in electron energy loss spectrometers 2–3 decades earlier. Two noteworthy examples of the types of STEM investigation that aberration correction has made possible are shown: imaging of single-atom impurities in graphene and analyzing atomic bonding of single atoms by electron energy loss spectroscopy (EELS). Looking towards the future, a new all-magnetic monochromator is described. The monochromator uses several of the principles pioneered in round lens aberration correction, and it employs stabilization schemes that make it immune to variations in the high voltage of the microscope and in the monochromator main prism current. Tests of the monochromator carried out at 60 keV have demonstrated energy resolution as good as 12 meV and monochromated probe size of ~1.2 Å. These results were obtained in separate experiments, but they indicate that the instrument can perform imaging and EELS with an atom-sized probe 〈30 meV wide in energy, and that an improvement in energy resolution to 10 meV and beyond should be possible in the future.
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 95
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    Development of Cs and Cc correctors for transmission electron microscopy (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: Novel spherical aberration (Cs) and chromatic aberration (Cc) correctors, which correct aberrations using a new principle, were developed. The asymmetric Cs correctors were designed for use in the probe- and image-forming systems at 300 kV to diminish undesired parasitic aberrations. The correctors composed of non-equivalent multipoles connecting with a demagnifying transfer doublet in the system. The axial aberrations were corrected well up to the fifth order except 6-fold astigmatism ( A 6 ) experimentally. Next, we developed superior Cs correctors for probe- and image-forming systems of low voltage microscope that uses triple dodecapoles to correct 6-fold astigmatism ( A 6 ). An important feature of this system is the rotation of the 3-fold astigmatism azimuth at the second dodecapole. The optimum rotation of the three hexapole fields for the compensation of A 6 was derived from theoretical calculations. The experimental results confirmed the compensation of A 6 and the third-order Cs. Finally, a unique Cc corrector, which utilized the concave lens effect formed by a long quadrupole field, was designed. The performance of the Cc corrector was investigated using a 30-kV transmission electron microscope. The results confirmed that Cc correction was achieved.
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 96
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    Problems in obtaining perfect images by single-particle electron cryomicroscopy of biological structures in amorphous ice (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: Theoretical considerations together with simulations of single-particle electron cryomicroscopy images of biological assemblies in ice demonstrate that atomic structures should be obtainable from images of a few thousand asymmetric units, provided the molecular weight of the whole assembly being studied is greater than the minimum needed for accurate position and orientation determination. However, with present methods of specimen preparation and current microscope and detector technologies, many more particles are needed, and the alignment of smaller assemblies is difficult or impossible. Only larger structures, with enough signal to allow good orientation determination and with enough images to allow averaging of many hundreds of thousands or even millions of asymmetric units, have successfully produced high-resolution maps. In this review, we compare the contrast of experimental electron cryomicroscopy images of two smaller molecular assemblies, namely apoferritin and beta-galactosidase, with that expected from perfect simulated images calculated from their known X-ray structures. We show that the contrast and signal-to-noise ratio of experimental images still require significant improvement before it will be possible to realize the full potential of single-particle electron cryomicroscopy. In particular, although reasonably good orientations can be obtained for beta-galactosidase, we have been unable to obtain reliable orientation determination from experimental images of apoferritin. Simulations suggest that at least 2-fold improvement of the contrast in experimental images at ~10 Å resolution is needed and should be possible.
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 97
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    Cover Page (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 98
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    Subscription Page (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 99
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    A review of fine structures of nanoporous materials as evidenced by microscopic methods (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: This paper reviews diverse capabilities offered by modern electron microscopy techniques in studying fine structures of nanoporous crystals such as zeolites, silica mesoporous crystals, metal organic frameworks and yolk-shell materials. For the case of silica mesoporous crystals, new approaches that have been developed recently to determine the three-dimensionally periodic average structure, e.g., through self-consistent analysis of electron microscope images or through consideration of accidental extinctions, are presented. Various structural deviations in nanoporous materials from their average structures including intergrowth, surface termination, incommensurate modulation, quasicrystal and defects are demonstrated. Ibidem observations of the scanning electron microscope and atomic force microscope give information about the zeolite-crystal-growth mechanism, and an energy for unstitching a building-unit from a crystal surface is directly observed by an anatomic force microscope. It is argued how these observations lead to a deeper understanding of the materials.
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
    Published by Oxford University Press on behalf of The Japanese Electron Microscopy Society.
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  • 100
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    Development of an environmental high-voltage electron microscope for reaction science (2013)
    Oxford University Press
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    Publication Date: 2013-02-19
    Description: Environmental transmission electron microscopy and ultra-high resolution electron microscopic observation using aberration correctors have recently emerged as topics of great interest. The former method is an extension of the so-called in situ electron microscopy that has been performed since the 1970s. Current research in this area has been focusing on dynamic observation with atomic resolution under gaseous atmospheres and in liquids. Since 2007, Nagoya University has been developing a new 1-MV high voltage (scanning) transmission electron microscope that can be used to observe nanomaterials under conditions that include the presence of gases, liquids and illuminating lights, and it can be also used to perform mechanical operations to nanometre-sized areas as well as electron tomography and elemental analysis by electron energy loss spectroscopy. The new instrument has been used to image and analyse various types of samples including biological ones.
    Print ISSN: 0022-0744
    Electronic ISSN: 1477-9986
    Topics: Electrical Engineering, Measurement and Control Technology , Natural Sciences in General , Physics
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