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  • 1
    Language: English
    In: Journal of Molecular Biology, 06 November 2015, Vol.427(22), pp.3527-3537
    Description: The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoining sarcomeres and transmits tension along myofibrils during muscular contraction. It is also the location of a number of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies. Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contraction and the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-section electron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinal sections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles have wider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously been used for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, we have used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extracted and compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links, presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres. The reconstruction shows that the terminal 5–7 nm of the actin filaments within the Z-band is devoid of any α-actinin links and is likely to be the location of capping protein CapZ.
    Keywords: Electron Tomography ; Electron Microscopy ; Z-Line ; Z-Disc ; Α-Actinin ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 2
    Language: English
    In: BBA - Molecular Cell Research, September 2015, Vol.1853(9), pp.2012-2017
    Description: Communication between organelles is a necessary consequence of intracellular compartmentalization. Membrane contact sites (MCSs) are regions where the membranes of two organelles come into close apposition allowing exchange of small molecules and ions including Ca . The ER, the cell's major Ca store, forms an extensive and dynamic network of contacts with multiple organelles. Here we review established and emerging roles of ER contacts as platforms for Ca exchange and further consider a potential role for Ca in the regulation of MCS formation. We additionally discuss the challenges associated with the study of MCS biology and highlight advances in microscopy-based solutions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
    Keywords: Calcium ; Membrane Contact Sites (Mcss) ; Ca2 + Exchange ; Biology ; Chemistry
    ISSN: 0167-4889
    E-ISSN: 1879-2596
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 29 December 2015, Vol.112(52), pp.15922-7
    Description: The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.
    Keywords: Disc Renewal ; Protocadherin ; Rhodopsin ; Rod Photoreceptors ; Cadherins -- Metabolism ; Cell Membrane -- Metabolism ; Photoreceptor Cells -- Metabolism ; Retinal Photoreceptor Cell Inner Segment -- Metabolism ; Retinal Rod Photoreceptor Cells -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: PLoS ONE, 01 January 2018, Vol.13(1), p.e0191048
    Description: Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 5
    Language: English
    In: Biochimica et biophysica acta, September 2015, Vol.1853(9), pp.2012-7
    Description: Communication between organelles is a necessary consequence of intracellular compartmentalization. Membrane contact sites (MCSs) are regions where the membranes of two organelles come into close apposition allowing exchange of small molecules and ions including Ca²⁺. The ER, the cell's major Ca²⁺ store, forms an extensive and dynamic network of contacts with multiple organelles. Here we review established and emerging roles of ER contacts as platforms for Ca²⁺ exchange and further consider a potential role for Ca²⁺ in the regulation of MCS formation. We additionally discuss the challenges associated with the study of MCS biology and highlight advances in microscopy-based solutions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
    Keywords: Ca2+ Exchange ; Calcium ; Er ; Membrane Contact Sites (Mcss) ; Calcium -- Metabolism ; Calcium Signaling -- Physiology ; Endoplasmic Reticulum -- Metabolism ; Intracellular Membranes -- Metabolism
    ISSN: 0006-3002
    ISSN: 01674889
    E-ISSN: 18792596
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 6
    Book
    Book
    United Kingdom: Andrews UK Ltd
    Language: English
    Description: Thomas Burgoyne and Norman Astley both claimed to have contacted members of the 'Brotherhood of Light', a group of advanced ethereal beings who attempt to guide the...
    Source: Dawsonera
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  • 7
    Language: English
    In: Journal of immunology (Baltimore, Md. : 1950), 15 May 2017, Vol.198(10), pp.4074-4085
    Description: Lymphocyte transendothelial migration (TEM) is critically dependent on intraendothelial signaling triggered by adhesion to ICAM-1. Here we show that endothelial MAPKs ERK, p38, and JNK mediate diapedesis-related and diapedesis-unrelated functions of ICAM-1 in cerebral and dermal microvascular endothelial cells (MVECs). All three MAPKs were activated by ICAM-1 engagement, either through lymphocyte adhesion or Ab-mediated clustering. MAPKs were involved in ICAM-1-dependent expression of TNF-α in cerebral and dermal MVECs, and CXCL8, CCL3, CCL4, VCAM-1, and cyclooxygenase 2 (COX-2) in cerebral MVECs. Endothelial JNK and to a much lesser degree p38 were the principal MAPKs involved in facilitating diapedesis of CD4 lymphocytes across both types of MVECs, whereas ERK was additionally required for TEM across dermal MVECs. JNK activity was critical for ICAM-1-induced F-actin rearrangements. Furthermore, activation of endothelial ICAM-1/JNK led to phosphorylation of paxillin, its association with VE-cadherin, and internalization of the latter. Importantly ICAM-1-induced phosphorylation of paxillin was required for lymphocyte TEM and converged functionally with VE-cadherin phosphorylation. Taken together we conclude that during lymphocyte TEM, ICAM-1 signaling diverges into pathways regulating lymphocyte diapedesis, and other pathways modulating gene expression thereby contributing to the long-term inflammatory response of the endothelium.
    Keywords: Signal Transduction ; Transendothelial and Transepithelial Migration ; Endothelial Cells -- Metabolism ; Inflammation -- Metabolism ; Intercellular Adhesion Molecule-1 -- Metabolism ; Mitogen-Activated Protein Kinases -- Metabolism ; P38 Mitogen-Activated Protein Kinases -- Metabolism
    ISSN: 00221767
    E-ISSN: 1550-6606
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  • 8
    In: Analytical Chemistry, Jan 15, 2001, Vol.73(2), p.192
    Description: Two fundamentally different approaches, termed "pointwise" and "peakwise," are currently used to correct hydrogen isotope ratio monitoring data for the presence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] ion contributions. Consideration of the underlying assumptions shows that the peakwise approach is valid only for peaks with the same functional shape and only when background signals do not vary. The pointwise correction is much more versatile and can be used even when peak shapes and sizes, as well as background signals, vary significantly. It is not exact and is limited in accuracy by (1) the signal-broadening effects of electronic time constants, (2) the analog-to-digital conversion frequency, and (3) the highest frequency of the sample signal. To minimize errors for typical gas chromatographic signals, time constants of [is less than] 500 ms and analog-to-digital sampling intervals of [is less than or equal to] 250 ms are needed. Errors are further minimized by matching sample and standard peaks in both amplitude and D/H ratio. Using the pointwise algorithm, we demonstrate that a series of 14 homologous n-alkanes varying in concentration over a 5-fold range can be analyzed with a mean precision of 2.3% and no systematic errors.
    Keywords: Analytical Chemistry -- Methods ; Ions -- Measurement ; Hydrogen Isotopes
    ISSN: 0003-2700
    Source: Cengage Learning, Inc.
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  • 9
    In: Analytical Chemistry, Jan 15, 2001, Vol.73(2), p.200
    Description: The [H.sub.3] factor, K, is a parameter required in high-precision, mass spectrometric analyses of hydrogen isotopic abundances. When H2 is used as the sample gas, R* = R - [Ki.sub.2], where R* is the true HD/[H.sub.2] ratio, R is the observed (mass 3)/(mass 2) ion-current ratio, and [i.sub.2] is the ion current at mass 2. Four different methods for the determination of K were defined and tested under conditions characteristic of isotope ratio monitoring systems. Three of these were peak-based. The fourth employed steady flows of [H.sub.2] from a conventional inlet system. Results obtained using the latter method were more precise (standard deviation of K = 0.1 versus ~0.6 ppm [mV.sup.-1] for the peak-based methods). However, use of the resulting values of K for correction of isotope ratio monitoring GC/MS results led to systematic errors as large as 9%, whereas use of the peak-based values led to no systematic errors. Values of K were only weakly dependent on the pressure of He, declining ~5% for each 10-fold increase in [P.sub.He]. Small variations in partial pressures of [H.sub.2]O and [CH.sub.4], potential contaminants under isotope ratio monitoring conditions, had no significant effect on values of K.
    Keywords: Mass Spectrometry -- Evaluation ; Hydrogen Isotopes
    ISSN: 0003-2700
    Source: Cengage Learning, Inc.
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  • 10
    Language: English
    In: BMC complementary and alternative medicine, 05 August 2015, Vol.15, pp.264
    Description: Arum palaestinum is a plant commonly found in the Middle East that is ingested as an herbal remedy to fight cancer. However, no studies have examined the direct effect of the plant/plant extract on tumor growth in an animal model. Verified prostate cancer cells were plated as 3D spheroids to determine the effect of extract from boiled Arum Palaestinum Boiss roots. In addition, male NU/NU mice (8 weeks old) with xenograft tumors derived from the prostate cancer cell line were treated daily with 1000 mg/kg body weight gavage of the suspension GZ17. The tumor growth was measured repeatedly with calipers and the excised tumors were weighed at the termination of the 3 week study. Control mice (10 mice in each group) received vehicle in the same manner and volume. The number of live prostate cancer cells declined in a dose/dependent manner with a 24 h exposure to the extract at doses of 0.015 to 6.25 mg/mL. A fortified version of the extract (referred to as GZ17) that contained higher levels of isovanillin, linolenic acid and β-sitosterol had a stronger effect on the cell death rate, shifting the percentage of dead cells from 30 % to 55 % at the highest dose while the vehicle control had no effect on cell numbers. When GZ17 was applied to non-cancer tissue, in this case, human islets, there was no cell death at doses that were toxic to treated cancer cells. Preliminary toxicity studies were conducted on rats using an up-down design, with no signs of toxic effect at the highest dose. NU/NU mice with xenograft prostate tumors treated with GZ17 had a dramatic inhibition of tumor progression, while tumors in the control group grew steadily through the 3 weeks. The rate of tumor volume increase was 73 mm(3)/day for the vehicle group and 24 mm(3)/day for the GZ17 treated mice. While there was a trend towards lower excised tumor weight at study termination in the GZ17 treatment group, there was no statistical difference. Fortified Arum palaestinum Boiss caused a reduction in live cells within prostate cancer spheroids and blocked tumor growth in xenografted prostate tumors in mice without signs of toxicity.
    Keywords: Antineoplastic Agents ; Benzaldehydes ; Plant Extracts ; Prostatic Neoplasms ; Sitosterols ; Alpha-Linolenic Acid ; Arum -- Chemistry
    E-ISSN: 1472-6882
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