• Prostaglandin production by melanocytic cells and the effect of a-melanocyte stimulating hormone

      Nicolaou, Anna; Estdale, Siân E.; Tsatmali, Marina; Herrero, Daniel Pascual; Thody, Anthony J. (2004)
      Prostaglandins are potent mediators of the inflam-matory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and-2) and thus have the capability to produce prostaglandins. TheFM55 cells produced predominantly PGE2and PGF2a, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, a-melanocyte stimulating hormone(a-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-a in the former. These results indicate that melanocytes produce prostaglandins and that a-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.
    • Prostaglandin-E2 is produced by adult human epidermal melanocytes in response to UVB in a melanogenesis-independent manner.

      Gledhill, Karl; Rhodes, L.E.; Brownrigg, M.; Haylett, A.K.; Masoodi, Mojgan; Thody, Anthony J.; Nicolaou, Anna; Tobin, Desmond J. (Wiley, 2010)
      Erythema occurs in human skin following excessive exposure to ultraviolet radiation (UVR), and this is in part mediated by the vasodilator prostaglandin E2 (PGE2). While keratinocytes are a major source of this pro-inflammatory eicosanoid, epidermal melanocytes (EM) also express some of the cellular machinery required for PGE2 production. The primary aim of this study is to determine whether EM can produce PGE2 and so potentially also contribute to UVR-induced skin inflammation. Furthermore, we investigate the likely pathway by which this PGE2 production is achieved and investigate whether PGE2 production by EM is correlated with melanogenic capacity. Primary cultures of EM were established from nine normal healthy individuals with skin phototype-1 (n=4) and 4 (n=5), and PGE2 production and melanogenic status were assessed. EM produced PGE2 under baseline conditions and this was increased further upon stimulation with arachidonic acid. Moreover, EM expressed cytoplasmic phospholipase A2, cyclooxygenase-1 and cytoplasmic prostaglandin E synthase. However, no EM culture expressed cyclooxygenase-2 under baseline conditions or following arachidonic acid, UVB- or H2O2 treatments. PGE2 production in response to UVB was highly variable in EM cultures derived from different donors but when pooled for skin phototype exhibited a positive correlation only with SPT-1 derived EM. Interestingly, PGE2 production by EM in response to UVB showed no correlation with baseline levels of melanin, tyrosinase expression/activity or tyrosinase-related protein-1 expression. However, there was an apparent negative correlation with baseline expression of dopachrome tautomerase (DCT), a melanogenic enzyme with reported anti-oxidant potential. These findings suggest that EM have the potential to contribute to UVR-induced erythema via PGE2 production, but that this response may be more related to oxidative stress than to their melanogenesis status.
    • The prostamide-related glaucoma therapy, bimatoprost, offers a novel approach for treating scalp alopecias

      Khidhir, Karzan Ghafur; Woodward, D.F.; Farjo, N.P.; Farjo, B.K.; Tang, E.S.; Wang, J.W.; Randall, Valerie A.; Picksley, Stephen M. (2013)
      Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F2α-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F2α analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.
    • Proteases in cancer drug delivery

      Vandooren, J.; Opdenakker, G.; Loadman, Paul M.; Edwards, D.R. (2016-02-01)
      Whereas protease inhibitors have been developed successfully against hypertension and viral infections, they have failed thus far as cancer drugs. With advances in cancer profiling we now better understand that the tumor “degradome” (i.e. the repertoire of proteases and their natural inhibitors and interaction partners) forms a complex network in which specific nodes determine the global outcome of manipulation of the protease web. However, knowing which proteases are active in the tumor micro-environment, we may tackle cancers with the use of Protease-Activated Prodrugs (PAPs). Here we exemplify this concept for metallo-, cysteine and serine proteases. PAPs not only exist as small molecular adducts, containing a cleavable substrate sequence and a latent prodrug, they are presently also manufactured as various types of nanoparticles. Although the emphasis of this review is on PAPs for treatment, it is clear that protease activatable probes and nanoparticles are also powerful tools for imaging purposes, including tumor diagnosis and staging, as well as visualization of tumor imaging during microsurgical resections.
    • Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells

      Fatokun, Amos A.; Tome, M.; Smith, R.A.; Darlington, L.G.; Stone, T.W. (2015-12)
      Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.
    • Protein deregulation associated with breast cancer metastasis

      Chan, K.K.; Matchett, K.B.; McEnhill, P.M.; Dakir, El-Habib; McMullin, M.F.; El-Tanani, Y.; Patterson, Laurence H.; Faheem, A.; Rudland, P.S.; McCarron, P.A.; et al. (2015-08)
      Breast cancer is one of the most prevalent malignancies worldwide. It consists of a group of tumor cells that have the ability to grow uncontrollably, overcome replicative senescence (tumor progression) and metastasize within the body. Metastases are processes that consist of an array of complex gene dysregulation events. Although these processes are still not fully understood, the dysregulation of a number of key proteins must take place if the tumor cells are to disseminate and metastasize. It is now widely accepted that future effective and innovative treatments of cancer metastasis will have to encompass all the major components of malignant transformation. For this reason, much research is now being carried out into the mechanisms that govern the malignant transformation processes. Recent research has identified key genes involved in the development of metastases, as well as their mechanisms of action. A detailed understanding of the encoded proteins and their interrelationship generates the possibility of developing novel therapeutic approaches. This review will focus on a select group of proteins, often deregulated in breast cancer metastasis, which have shown therapeutic promise, notably, EMT, E-cadherin, Osteopontin, PEA3, Transforming Growth Factor Beta (TGF-β) and Ran.
    • Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

      Heathcote, H.R.; Mancini, S.J.; Strembitska, A.; Jamal, K.; Reihill, J.A.; Palmer, Timothy M.; Gould, G.W.; Salt, I.P. (2016)
      The key metabolic regulator, AMP-activated protein kinase (AMPK) is reported to be downregulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491 respectively as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.
    • A proteomic approach to the identification of cytochrome P450 isoforms in male and female rat liver by nanoscale liquid chromatography-electrospray ionization-tandem mass spectrometry.

      Nisar, S.; Lane, C.S.; Wilderspin, A.F.; Welham, K.J.; Griffiths, W.J.; Patterson, Laurence H. (American Society for Pharmacology and Experimental Therapeutics., 2004)
      Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.
    • A proteomic investigation of Streptococcus agalactiae grown under conditions associated with neonatal exposure reveals the upregulation of the putative virulence factor C protein β antigen

      Yang, Q.; Zhang, M.; Harrington, Dean J.; Black, G.W.; Sutcliffe, I.C. (2010-06)
      Streptococcus agalactiae is a major neonatal pathogen that is able to adapt to a variety of host environments, including both rectal and vaginal maternal carriage, growth in amniotic fluid and at various neonatal body sites. As such it is important to elucidate the patterns of protein expression that are associated with S. agalactiae growth under these different in vivo conditions. To this end, we have grown S. agalactiae strain A909 under in vitro conditions reflecting those associated with maternal vaginal carriage (low pH, low oxygen, nutrient stress) and those associated with exposure to body fluids during invasive disease (neutral pH, aeration, nutrient sufficient). The protein profiles of bacterial cells grown under each of these conditions were compared using a proteome approach. A total of 76 proteins were reproducibly identified 16 of which were shown to be differentially expressed. The putative virulence factor C protein β and several proteins linked to resistance to oxidative stress were found to be upregulated under the conditions hypothesised to reflect those associated with foetal exposure to S. agalactiae. Thus, these data add to the currently limited understanding of the molecular basis of S. agalactiae GBS adaptation to different environmental conditions.
    • A proteomic investigation of Streptococcus agalactiae reveals that human serum induces the C protein β antigen and arginine deiminase

      Yang, Q.; Zhang, M.; Harrington, Dean J.; Black, G.W.; Sutcliffe, I.C. (2011-08)
      Streptococcus agalactiae is a major neonatal pathogen. Disease progression is characterised by bacterial adaptation from commensal maternal vaginal colonisation to environments associated with neonatal disease, including exposure to blood. To explore this adaptation in vitro, we have used proteomics to identify proteins differentially expressed following growth on Todd Hewitt agar in the presence or absence of 10% v/v human serum. Twelve differentially expressed proteins were identified. Notably, the C protein β antigen and arginine deiminase proteins were upregulated following growth in the presence of human serum, consistent with previous studies implicating these two proteins in the pathogenesis of S. agalactiae disease.
    • Proteomics of tissue factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control

      Lento, S.; Brioschi, M.; Barcella, S.; Nasim, Md. Talat; Ghilardi, S.; Barbieri, S.S.; Tremoli, E.; Banfi, C. (2015-04-24)
      It has long been known that Tissue Factor (TF) plays a role in blood coagulation and has a direct thrombotic action that is closely related to cardiovascular risk, but it is becoming increasingly clear that it has a much wider range of biological functions that range from inflammation to immunity. It is also involved in maintaining heart haemostasis and structure, and the observation that it is down-regulated in the myocardium of patients with dilated cardiomyopathy suggests that it influences cell-to-cell contact stability and contractility, and thus contributes to cardiac dysfunction. However, the molecular mechanisms underlying these coagulation-independent functions have not yet been fully elucidated. In order to analyse the influence of TF on the cardiomyocitic proteome, we used functional biochemical approaches incorporating label-free quantitative proteomics and gene silencing, and found that this provided a powerful means of identifying a new role for TF in regulating splicing machinery together with the expression of several proteins of the spliceosome, and mRNA metabolism with a considerable impact on cell viability.
    • Protocol for sectioning human dentine: expanded from Methods 1 and 2

      Beaumont, Julia; Gledhill, Andrew R.; Lee-Thorp, Julia A.; Montgomery, Janet (2013)
    • Proton location in acid⋯pyridine hydrogen bonds of multi-component crystals

      Seaton, Colin C. (2014)
      The design of new functional crystalline materials requires an understanding of the factors that control salt and co-crystal formation. These states often only differ in the location of the proton and are influenced by chemical and crystallographic factors. The interaction between a carboxylic acid and a pyridine is a frequently used supramolecular synthon in crystal engineering which can exist as either a co-crystal (CO2H⋯N) or salt (CO2−⋯HN+). The results of a Cambridge Structure Database search indicate that the nature of the functional groups on the pyridine play a stronger role in selection of the phase than those of the acid. However, the nature of the local hydrogen bonding of the interaction also adjusts the potential for proton transfer. This was demonstrated by ab initio modelling of the energy landscape for binary and ternary co-crystals by inclusion of varying components of the local environment.
    • Proton transfer and hydrogen bonding in the organic solid state: a combined XRD/XPS/ssNMR study of 17 organic acid–base complexes

      Stevens, J.S.; Byard, S.J.; Seaton, Colin C.; Sadiq, G.; Davey, R.J.; Schroeder, S.L.M. (2014-01-21)
      The properties of nitrogen centres acting either as hydrogen-bond or Brønsted acceptors in solid molecular acid–base complexes have been probed by N 1s X-ray photoelectron spectroscopy (XPS) as well as 15N solid-state nuclear magnetic resonance (ssNMR) spectroscopy and are interpreted with reference to local crystallographic structure information provided by X-ray diffraction (XRD). We have previously shown that the strong chemical shift of the N 1s binding energy associated with the protonation of nitrogen centres unequivocally distinguishes protonated (salt) from hydrogen-bonded (co-crystal) nitrogen species. This result is further supported by significant ssNMR shifts to low frequency, which occur with proton transfer from the acid to the base component. Generally, only minor chemical shifts occur upon co-crystal formation, unless a strong hydrogen bond is formed. CASTEP density functional theory (DFT) calculations of 15N ssNMR isotropic chemical shifts correlate well with the experimental data, confirming that computational predictions of H-bond strengths and associated ssNMR chemical shifts allow the identification of salt and co-crystal structures (NMR crystallography). The excellent agreement between the conclusions drawn by XPS and the combined CASTEP/ssNMR investigations opens up a reliable avenue for local structure characterization in molecular systems even in the absence of crystal structure information, for example for non-crystalline or amorphous matter. The range of 17 different systems investigated in this study demonstrates the generic nature of this approach, which will be applicable to many other molecular materials in organic, physical, and materials chemistry.
    • Proximity Effects in the Electron Impact Mass Spectra of 2-Substituted Benzazoles

      Chantler, Thomas; Perrin, Victoria L.; Donkor, Rachel E.; Cawthorne, Richard S.; Bowen, Richard D. (2004)
      The 70 eV electron impact mass spectra of a wide range of 2-substituted benzazoles are reported and discussed. Particular attention is paid to the mechanistic significance and analytical utility of [M–H]+ and [M–X]+ signals in the spectra of benzazoles in which the 2-substituent contains a terminal aryl group with one or more substituents, X. Loss of H or X occurs preferentially from an ortho-position from ionized 2-benzylbenzimidazoles, 2-phenethylbenzimidazoles, 2-styrylbenzimidazoles, 2-styrylbenzoxazoles and 2-styrylbenzothiazoles. In the three styrylbenzazole series, the [M–H]+ and/or [M–X]+ signals dominate the spectra. This unusually facile loss of H or X may be attributed to a proximity effect, in which cyclization of the ionized molecule is followed by elimination of an ortho-substituent to give an exceptionally stable polycyclic ion. Formation of a new five- or six-membered ring by the proximity effect occurs rapidly; cyclization to a seven-membered ring takes place rather less readily; but formation of a ring with only four atoms or more than seven atoms is not observed to a significant extent. The proximity effect competes effectively with loss of a methyl radical by simple cleavage of an ethyl, isopropyl and even a t-butyl group in the pendant aromatic ring of ionized 2-(4-alkylstyryl) benzazoles.
    • Pseudo electron-deficient organometallics: limited reactivity towards electron-donating ligands

      Pitto-Barry, Anaïs; Lupan, A.; Zegke, Markus; Swift, Thomas; Attia, A.A.A.; Lord, Rianne M.; Barry, Nicolas P.E. (2017)
      Half-sandwich metal complexes are of considerable interest in medicine, material, and nanomaterial chemistry. The design of libraries of such complexes with particular reactivity and properties is therefore a major quest. Here, we report the unique and peculiar reactivity of eight apparently 16-electron half-sandwich metal (ruthenium, osmium, rhodium, and iridium) complexes based on benzene-1,2-dithiolato and 3,6-dichlorobenzene-1,2-dithiolato chelating ligands. These electron-deficient complexes do not react with electron-donor pyridine derivatives, even with the strong σ-donor 4-dimethylaminopyridine (DMAP) ligand. The Ru, Rh, and Ir complexes accept electrons from the triphenylphosphine ligand (σ-donor, π-acceptor), whilst the Os complexes were found to be the first examples of non-electron-acceptor electron-deficient metal complexes. We rationalized these unique properties by a combination of experimental techniques and DFT/TDFT calculations. The synthetic versatility offered by this family of complexes, the low reactivity at the metal center, and the facile functionalization of the non-innocent benzene ligands is expected to allow the synthesis of libraries of pseudo electron-deficient half-sandwich complexes with unusual properties for a large range of applications.
    • Pseudomonas aeruginosa: a formidable and ever-present adversary.

      Kerr, Kevin G.; Snelling, Anna M. (2009)
      Pseudomonas aeruginosa is a versatile pathogen associated with a broad spectrum of infections in humans. In healthcare settings the bacterium is an important cause of infection in vulnerable individuals including those with burns or neutropenia or receiving intensive care. In these groups morbidity and mortality attributable to P. aeruginosa infection can be high. Management of infections is difficult as P. aeruginosa is inherently resistant to many antimicrobials. Furthermore, treatment is being rendered increasingly problematic due to the emergence and spread of resistance to the few agents that remain as therapeutic options. A notable recent development is the acquisition of carbapenemases by some strains of P. aeruginosa. Given these challenges, it would seem reasonable to identify strategies that would prevent acquisition of the bacterium by hospitalised patients. Environmental reservoirs of P. aeruginosa are readily identifiable, and there are numerous reports of outbreaks that have been attributed to an environmental source; however, the role of such sources in sporadic pseudomonal infection is less well understood. Nevertheless there is emerging evidence from prospective studies to suggest that environmental sources, especially water, may have significance in the epidemiology of sporadic P. aeruginosa infections in hospital settings, including intensive care units. A better understanding of the role of environmental reservoirs in pseudomonal infection will permit the development of new strategies and refinement of existing approaches to interrupt transmission from these sources to patients.
    • Pseudostatic and dynamic nanomechanics of the tunica adventitia in elastic arteries using atomic force microscopy

      Grant, Colin A.; Twigg, Peter C. (2013)
      Tunica adventitia, the outer layer of blood vessels, is an important structural feature, predominantly consisting of collagen fibrils. This study uses pseudostatic atomic force microscopy (AFM) nanoindentation at physiological conditions to show that the distribution of indentation modulus and viscous creep for the tunica adventitia of porcine aorta and pulmonary artery are distinct. Dynamic nanoindentation demonstrates that the viscous dissipation of the tunica adventitia of the aorta is greater than the pulmonary artery. We suggest that this mechanical property of the aortic adventitia is functionally advantageous due to the higher blood pressure within this vessel during the cardiac cycle. The effects on pulsatile deformation and dissipative energy losses are discussed.
    • A Psychophysical Evaluation of Inverse Tone Mapping Techniques.

      Banterle, F.; Ledda, P.; Debattista, K.; Bloj, Marina; Artussi, A.; Chalmers, A. (2009)
      In recent years inverse tone mapping techniques have been proposed for enhancing low-dynamic range (LDR) content for a high-dynamic range (HDR) experience on HDR displays, and for image based lighting. In this paper, we present a psychophysical study to evaluate the performance of inverse (reverse) tone mapping algorithms. Some of these techniques are computationally expensive because they need to resolve quantization problems that can occur when expanding an LDR image. Even if they can be implemented efficiently on hardware, the computational cost can still be high. An alternative is to utilize less complex operators; although these may suffer in terms of accuracy. Our study investigates, firstly, if a high level of complexity is needed for inverse tone mapping and, secondly, if a correlation exists between image content and quality. Two main applications have been considered: visualization on an HDR monitor and image-based lighting.
    • Public participation and policy: unpacking connections in one British LA21

      Sharp, Liz (2002)
      Within western cultures, the term `public participation¿ has strong positive connotations, and is associated with the promotion of democracy. The contention of this paper is that these invocations of democracy - although not entirely inaccurate - obscure the varied and tangible effects of public participation on wider policy processes. Drawing on Sharp and Connelly 2001, this paper argues that participation should not be analysed in terms of the type of democracy it invokes, but rather in terms of the extent and nature of its influence on the policy process. In particular, the policy process is examined for conflict between participants over (1) the extent of participation, (2) the nature of participation and (3) the influence of the participation, as well as (4) the outcomes to which it leads. This approach to the analysis of participation is demonstrated through a study of one element of participation in an authority¿s Local Agenda 21 process. The paper concludes that participation is inherently political and practitioners need to act strategically to manage participation in support of progressive agendas.