• Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate.

      Cooksey, C J; Garratt, P J; Land, Edward J; Ramsden, C A; Riley, P A; Department of Chemistry, Christopher Ingold Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ, UK. (1998-08-01)
      When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1.cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol.min-1 and the apparent Km was 374 microM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol.min-1 and Km 309 microM. The quinomethane was found to decay with a rate constant of 2k 38 M-1.s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.
    • Tyrosinase-mediated cytotoxicity of 4-substituted phenols: quantitative structure-thiol-reactivity relationships of the derived o-quinones.

      Cooksey, C J; Land, Edward J; Ramsden, C A; Riley, P A; Department of Chemistry, University College London, UK. (1995-03)
      Rate constants have been determined for reactions between biologically significant thiols, represented by cysteine and glutathione, and a series of 10 4-substituted o-quinones, and unsubstituted o-quinone itself, generated by rapid disproportionation of the semiquinones formed from the corresponding catechols by pulse radiolysis. The quantitative structure-reactivity relationships were investigated by examining the correlation between the rate constants and various Hammett and other parameters characterizing the electronic nature of the substituents. From these relationships, it can be concluded that the o-quinone reactivity with thiols increases with the electron-withdrawing capacity of the substituent groups and that this effect is principally due to resonance effects. Such relationships allow the prediction of likely reactivities with cellular thiols of further o-quinones whose 4-substituents have known electronic parameters. These reactivities are likely to be one of the critical factors determining overall cytotoxicity, assisting in the choice of improved melanogenesis-targeted anti-melanoma drugs.
    • Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast.

      Wilkinson, Caroline R M; Dittmar, Gunnar A G; Ohi, Melanie D; Uetz, Peter; Jones, Nic; Finley, Daniel; Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. cwilkinson@picr.man.ac.uk (2004-12-29)
      Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.
    • Ubiquitin-like proteins in the DNA damage response: the next generation

      Da Costa, Isabelle C; Schmidt, Christine K; Manchester Cancer Research Centre, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, 555 Wilmslow Road, Manchester (2020)
      DNA suffers constant insult from a variety of endogenous and exogenous sources. To deal with the arising lesions, cells have evolved complex and coordinated pathways, collectively termed the DNA damage response (DDR). Importantly, an improper DDR can lead to genome instability, premature ageing and human diseases, including cancer as well as neurodegenerative disorders. As a crucial process for cell survival, regulation of the DDR is multi-layered and includes several post-translational modifications. Since the discovery of ubiquitin in 1975 and the ubiquitylation cascade in the early 1980s, a number of ubiquitin-like proteins (UBLs) have been identified as post-translational modifiers. However, while the importance of ubiquitin and the UBLs SUMO and NEDD8 in DNA damage repair and signalling is well established, the roles of the remaining UBLs in the DDR are only starting to be uncovered. Herein, we revise the current status of the UBLs ISG15, UBL5, FAT10 and UFM1 as emerging co-regulators of DDR processes. In fact, it is becoming clear that these post-translational modifiers play important pleiotropic roles in DNA damage and/or associated stress-related cellular responses. Expanding our understanding of the molecular mechanisms underlying these emerging UBL functions will be fundamental for enhancing our knowledge of the DDR and potentially provide new therapeutic strategies for various human diseases including cancer.
    • Ubiquitin-like proteins: meet the family.

      Wilkinson, Caroline R M; Paterson Institute for Cancer research, Christie Hospital, Manchester, UK. cwilkinson@picr.man.ac.uk (2004-04)
    • The Ubiquitin/UBL drug target repertoire

      Osborne, Hugh C; Irving, E.; Schmidt, Christine K; Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Cancer Research Centre, Manchester M20 4GJ, (2020)
    • UHRF1-mediated tumor suppressor gene inactivation in nonsmall cell lung cancer.

      Daskalos, A; Oleksiewicz, U; Filia, A; Nikolaidis, G; Xinarianos, G; Gosney, J; Malliri, Angeliki; Field, J; Liloglou, T; Roy Castle Lung Cancer Research Programme, University of Liverpool, Department of Clinical and Molecular Cancer Medicine, Liverpool, United Kingdom. (2011-03-01)
      The UHRF1 gene possesses an essential role in DNA methylation maintenance, but its contribution to tumor suppressor gene hypermethylation in primary human cancers currently remains unclear.
    • UKEMS collaborative genotoxicity trial. Cytogenetic tests of 4CMB, BC and 4HMB: summary and appraisal.

      Scott, David; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (Great Britain) (1982)
    • Ultrastructural and morphometric alterations in bone marrow stromal tissue after 7 Gy irradiation.

      Yamazaki, K; Allen, Terence D; Department of Pathology, School of Medicine, Keio University, Tokyo, Japan. (1991)
      To evaluate the response of marrow stroma to 7 Gy irradiation, femoral bone marrow was fixed by vascular perfusion (so as to avoid the artificial destruction of sinus endothelia), and was examined using light and electron microscopy with morphometric methods. The radiation caused a marked decrease in hematopoietic cell number (NHC) within 3 days post-irradiation, followed by total recovery of hematopoiesis, which occurred gradually over 28 days. An increased number of fat cells was seen by 7 days. During the whole course of hypoplasia and recovery, the continuity of sinus wall, three-dimensional reticular mesh work in hematopoietic parenchyma, gap junctions (GJ) between stromal cells, the adventitial cell cover of sinus wall (ACC), and the stromal cell numbers of reticular cells (RC), sinus endothelia (SE), and macrophages (MP) were maintained. The cellularity of stromal components of RC, SE, and MP seemed passively increased in contrast to a reduction in numbers of NHC. A similar tendency was observed (1) between NHC and ACC and (2) between GJ and the cellularity of fat cells, which had a statistical significant correlation (p less than 0.05; t-test). The mechanism of radio resistance in bone marrow stroma and the possible functional adaptation and cellular coordination after irradiation are discussed.
    • Ultrastructural aspects of erythropoietic differentiation in long-term bone marrow culture.

      Allen, Terence D; Dexter, T Michael; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester, M20 9BX, UK (1982)
      Long-term liquid cultures of mouse bone marrow produce stem cell (CFU-S) and differentiated granulocytes for many months. Addition of AMS (anaemic mouse serum) to the cultures almost entirely eliminates the granulopoietic activity and stimulates erythropoiesis, with full erythroid maturation and the production of adult haemoglobin. Ultrastructural analysis of in situ fixed material reveals the cell shape and surface morphology of the erythroid maturation series, and the generation of erythroblastic islands in vitro. Each erythroblastic island consists of one or more synchronously maturing cohorts of erythroid cells undergoing four or five divisions between proerythroblast and normoblast. Each island is centered on a macrophage, which interacts with the developing erythroid population in several ways. Expelled nuclei are phagocytosed by te macrophage, which also has large areas of closely apposed membrane with the erythroid cells, gap junctions, and possible reciprocal vesicular activity. Changes in the adherent layer (stromal cells) also occur with the transition from granulopoiesis to erythropoiesis. There is a reduction in the endothelial cell cover, and mobilisation of lipid from the granulopoietic associated apidocytes.
    • Ultrastructural changes in Osmunda regalis Prothalli induced by X-irradiation.

      Allen, Terence D; Haigh, M V; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1973-01)
    • Ultrastructural morphology of three-dimensional colonies of cells derived from a hepatocellular carcinoma.

      Allen, Terence D; Iype, P; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979)
      Cultured hepatocellular carcinoma cells were studied during anchorage-independent growth in semi solid medium (Methocel). The regular occurrence of mitotic figures both at the surface and within the colonies precludes the possibility of such colonies being formed by re-aggregation. The estimated population doubling time in the three-dimensional (3-D) colonies is consistent with those two-dimensional of (2-D) colonies. Structures resembling bile canaliculi were observed between the closely opposed membranes from the well packed adjacent cells. Cell surface and ultrastructural features of the colonies and individual cells are presented and comparisons made with 2-D growth of normal and malignant liver cells in vitro. The formation of 3-D colonies may not only be an assay for transformed cells but also for predicting the type of tumors produced by re-innoculation of the in vitro transformed cells.
    • Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: the "neuro-reticular complex".

      Yamazaki, K; Allen, Terence D; Department of Pathology, School of Medicine, Keio University, Tokyo, Japan. (1990-03)
      In order to extend our understanding of the role of nerve fibers in the structure and function of bone marrow stroma, we have examined nerve terminals, arterioles, and capillaries in femoral bone marrow tissues of 50 C57BL strain mice, using electron microscopy and morphometric methods. Within the adventitia of arterioles, a particular type of cell, termed periarterial adventitial (PAA) cell, is characterized by a thin veil-like cytoplasm which concentrically surrounds both nerves and arterioles. Nerve fibers containing both unmyelinated and myelinated axons are distributed mainly between the layers of PAA cells, but are found rarely on the sinus walls or within the hematopoietic parenchyma. Quantitatively, the efferent nerve terminals with many synaptic vesicles are distributed mainly beside arterial smooth muscle cells (Type I: 58.8%) or between the layers of PAA cells (Type III: 33.2%), and rarely in hematopoietic parenchyma (Type II: 5.3%) or on sinus walls (Type IV: 2.7%). In the case of Type II-IV nerve terminals, efferent (autonomic) nerves and bone marrow stromal cells which are connected by gap junctions (sinus adventitial reticular cells, intersinusoidal reticular cells, and PAA cells) appear to constitute a potential functional unit for signal conduction. We would like to propose a new term for this anatomical unit in marrow, the "neuro-reticular complex."
    • Ultrastructural site variations in mouse epidermal organization.

      Allen, Terence D; Potten, Christopher S; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1976-07)
      Mouse dorsal, ear, tail and foot epidermis are compared according to their tissue architecture and cell kinetics. Cell proliferation is expected in terms of the daily volume of keratin replaced. The stratum corneum may be organized into vertical columns of squames, which may have minimal overlap as in dorsum and ear, or maximal overlap as in tail. Individual areas are adapted to their function both in squame fine structure and rate of cell replacement. The surface keratin loss/replacement rate is at its highest in foot and tail, and lowest in ear and dorsum. Observations on hairless mouse dorsum are also included.
    • Ultrastructure of cell loss in intestinal mucosa.

      Potten, Christopher S; Allen, Terence D; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1977-08)
    • Ultrastructure of giant plastids in a radiation induced mutant of Osmunda regalis.

      Allen, Terence D; Haigh, M V; Howard, Alma; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1973-03)
    • Ultraviolet light and melanoma.

      Craig, Sarah; Earnshaw, C; Virós, Amaya; Skin Cancer and Ageing Laboratory, CRUK Manchester Institute, The University of Manchester, UK (2018-01-30)
      Melanoma is a clinically heterogeneous disease, and current strategies for treatment of the primary tumour are based on pathological criteria alone. In the recent past, several DNA-sequencing and RNA-sequencing studies of primary and advanced melanoma samples have identified unique relationships between somatic mutations, genomic aberrations, and the genetic fingerprint of ultraviolet radiation (UVR). The recurrent patterns of genomic alterations reveal different disease pathways, drug targets and mechanisms limiting drug response. Here, we examine the known associations between the molecular categories of melanoma and the multidimensional UVR damage. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
    • Ultraviolet light-induced collagen degradation inhibits melanoma invasion

      Budden, Timothy; Gaudy, C.; Nagore, E; Viros, Amaya; Cancer Research UK Manchester Institute, Nether Alderley, Macclesfield, (2021)
      Ultraviolet radiation (UVR) increases the incidence of cutaneous melanoma. The ageing, sunexposed dermis accumulates UVR damage, and older patients develop more melanomas at UVR-exposed sites. As fibroblasts play key roles in the stromal response to UVR and in cancer progression, we investigated how long term UVR modifies dermal fibroblast function and how this affects melanoma invasion. Chronic UVR exposure on dermal fibroblasts showed that extracellular matrix pathways, particularly those involved in collagen catabolism, were upregulated in the absence of acute UVR. Importantly, the expression of collagen-cleaving matrix metalloprotein-1 (MMP1) was persistently upregulated. This resulted in persistent degradation of collagen 1, and an overall degraded and disorganised matrix. Collagen degradation by MMP1 decreased melanoma invasion in vitro. Conversely, both inhibiting extracellular matrix degradation and MMP1, or higher collagen 1 expression, restored the invasion of melanoma through collagen. Primary cutaneous melanomas of aged humans confirmed these in vitro findings, revealing significantly fewer cancer cells invade as single cells at the invasive front of melanomas arising in chronic sun damaged skin. We show high collagen deposition and melanoma cell invasion in the dermis are robust predictors of poor melanoma-specific survival in 3, international cohorts of primary melanoma. Thus, melanomas arising over UVR-damaged, collagen-poor skin are less invasive, and this reduced invasion improves survival. However, we discovered a subset of melanomas arising over collagen-poor, UVR-damaged dermis have a poor outcome, and found that increased new collagen synthesis by melanoma-associated fibroblasts at the invasive front in these cases restores melanoma single cell invasion and drives poor outcome. Finally, we demonstrate high COL1A1 gene expression is an early stage biomarker of poor outcome across a broad range of primary cancers.
    • Ultraviolet light-induced collagen degradation inhibits melanoma invasion

      Budden, Timothy; Gaudy-Marqueste, C.; Porter, Andrew P; Kay, E.; Gurung, Shilpa; Earnshaw, Charles; Roeck, K.; Craig, S.; Traves, V.; Krutmann, J.; et al. (2021)
      Ultraviolet radiation (UVR) damages the dermis and fibroblasts; and increases melanoma incidence. Fibroblasts and their matrix contribute to cancer, so we studied how UVR modifies dermal fibroblast function, the extracellular matrix (ECM) and melanoma invasion. We confirmed UVR-damaged fibroblasts persistently upregulate collagen-cleaving matrix metalloprotein-1 (MMP1) expression, reducing local collagen (COL1A1), and COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, inhibiting ECM degradation and MMP1 expression restored melanoma invasion. Primary cutaneous melanomas of aged humans show more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen, and collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. Thus, primary melanomas arising over collagen-degraded skin are less invasive, and reduced invasion improves survival. However, melanoma-associated fibroblasts can restore invasion by increasing collagen synthesis. Finally, high COL1A1 gene expression is a biomarker of poor outcome across a range of primary cancers.
    • Ultraviolet radiation-induced DNA damage is prognostic for outcome in melanoma.

      Trucco, Lucas D; Mundra, Piyushkumar A; Hogan, Kate; Garcia-Martinez, Pablo; Viros, Amaya; Mandal, Amit Kumar; Macagno, N; Gaudy-Marqueste, C; Allan, D; Baenke, Franziska; et al. (2018)
      Erratum in Publisher Correction: Ultraviolet radiation-induced DNA damage is prognostic for outcome in melanoma. [Nat Med. 2018] Abstract The melanoma genome is dominated by ultraviolet radiation (UVR)-induced mutations. Their relevance in disease progression is unknown. Here we classify melanomas by mutation signatures and identify ten recurrently mutated UVR signature genes that predict patient survival. We validate these findings in primary human melanomas; in mice we show that this signature is imprinted by short-wavelength UVR and that four exposures to UVR are sufficient to accelerate melanomagenesis.