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  • 1
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    Structure-based design of a streptavidin mutant specific for an artificial biotin analogue (2015)
    Oxford University Press
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    Publication Date: 2015-05-28
    Description: For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity ( K d 〈 10 –10 M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3a S ,4 S ,6a R )-2-iminohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a K d value of 5.9 x 10 –7 M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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  • 2
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    Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins (2015)
    Oxford University Press
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    Publication Date: 2015-09-30
    Description: The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell–cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pull-down assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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  • 3
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    RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility (2015)
    Oxford University Press
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    Publication Date: 2015-10-15
    Description: Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA II enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae . We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA II , rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae . Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA II in vivo , leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA II activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA II , thereby facilitating TEL binding to the ribosome.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression (2015)
    Oxford University Press
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    Publication Date: 2015-05-20
    Description: Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present s ingle- c ell mRNA 3 -prime end seq uencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.
    Keywords: Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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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.
    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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  • 6
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    Ca2+ enrichment in culture medium potentiates effect of oligonucleotides (2015)
    Oxford University Press
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    Publication Date: 2015-10-31
    Description: Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo . We show that Ca 2+ enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ~100 nm in size are found in Ca 2+ -enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles ( ~ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.
    Keywords: Targeted inhibition of gene function, DNA-Mediated Cell Transformation and Nucleic Acids Transfer
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c (2012)
    Oxford University Press
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    Publication Date: 2012-10-30
    Description: Through their ubiquitin ligase activity, Cbl-family proteins suppress signalling mediated by protein-tyrosine kinases (PTKs), but can also function as adaptor proteins to positively regulate signalling. The tyrosine kinase binding (TKB) domain of this family is critical for binding with tyrosine-phosphorylated target proteins. Here, we analysed the crystal structure of the TKB domain of Cbl-c/Cbl-3 (Cbl-c TKB), which is a distinct member of the mammalian Cbl-family. In comparison with Cbl TKB, Cbl-c TKB showed restricted structural flexibility upon phosphopeptide binding. A mutation in Cbl-c TKB augmenting this flexibility enhanced its binding to target phosphoproteins. These results suggest that proteins, post-translational modifications or mutations that alter structural flexibility of the TKB domain of Cbl-family proteins could regulate their binding to target phosphoproteins and thereby, affect PTK-mediated signalling.
    Print ISSN: 0021-924X
    Electronic ISSN: 1756-2651
    Topics: Biology , Chemistry and Pharmacology
    Published by Oxford University Press on behalf of The Japanese Biochemical Society (JBS).
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  • 8
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    In situ observation of structural transformation of gold nanorods under pulsed laser irradiation in an HVEM (2014)
    Oxford University Press
    Details
    Publication Date: 2014-08-03
    Description: A pulsed laser light illumination system was attached to a high-voltage electron microscope (HVEM) for in situ observation of light-induced behaviors of nano objects. The wavelength of emitted laser pulses was 1064, 532 or 266 nm, and the pulse duration was 6–8 ns. Using this combined HVEM system, we observed the deformation behavior of gold nanorods irradiated by a pulsed laser ( = 1064 nm) at an intensity of 310 J m –2 pulse or higher. A single shot of pulsed laser reduced the aspect ratio of the gold nanorods from 5 to a much smaller value. The extent of the reduction increased at higher laser intensities. However, at 310 J m –2 pulse –1 , additional pulsed shots induced limited further deformation. The mean aspect ratio approximated to 2.5 even after irradiation with 7 pulses (total fluence exceeding 2 MJ m –2 ). In situ high resolution transmission electron microscopy (HRTEM) observation revealed that deformation was accompanied by total atomic restructuring of the nanorod interiors.
    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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  • 9
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    Multislice simulation of transmission electron microscopy imaging of helium bubbles in Fe (2012)
    Oxford University Press
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    Publication Date: 2012-12-13
    Description: Formation of nanoscale helium (He) bubbles in reduced activation ferritic/martensitic steels may lead to degradation of mechanical properties of materials. Transmission electron microscopy (TEM) has commonly been used to image the Fresnel contrast of He bubbles, using an underfocus of 0.5–1 µm. This paper presents our study of multislice simulation of the size correlation between imaged Fresnel rings and the actual He bubbles. It was found that for bubbles equal to or 〉3 nm in diameter, the imaged bubble size, represented by its inner diameter of the first dark Fresnel ring ( D in ) in underfocused imaging conditions, increases with increasing electron-beam incoherency, but decreases with increasing underfocus. The electron-beam accelerating voltage, bubble size, bubble position and TEM sample thickness were found to have no significant influence on the deviation of D in from the actual bubble size ( D 0 ). However, for bubbles equal to or 〈2 nm, D in / D 0 increases dramatically with increasing underfocus when it is above a threshold limit (e.g. f  = –1 µm for a 2-nm bubble). The results of this study also suggested that He bubbles can be differentiated from argon (Ar) bubbles by contrast differences.
    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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  • 10
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    A method for accurate temperature measurement using infrared thermal camera (2012)
    Oxford University Press
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    Publication Date: 2012-08-07
    Description: The temperature distribution on a centre-holed thin foil of molybdenum, used as a sample and heated using a sample-heating holder for electron microscopy, was measured using an infrared thermal camera. The temperature on the heated foil area located near the heating stage of the heating holder is almost equal to the temperature on the heating stage. However, during the measurement of the temperature at the edge of the hole of the foil located farthest from the heating stage, a drop in temperature should be taken into consideration; however, so far, no method has been developed to locally measure the temperature distribution on the heated sample. In this study, a method for the accurate measurement of temperature distribution on heated samples for electron microscopy is discussed.
    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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