School of biomedical sciences
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The role of extracellular signal-regulated protein kinase (ERK) in -adrenoceptor-mediated vasodilatation in porcine isolated coronary artery Uhiara EO, Alexander SPHA, Roberts RE School of Biomedical Sciences, University of Nottingham -Adrenoceptor (-AR)-mediated vasodilatation is known to involve a number of components, including cAMP-dependent protein kinase, nitric oxide and potassium channels (Cardillo et al., 1997; White et al. 2001) In cultured cells expressing -ARs, isoprenaline and other -AR ligands increase extracellular signal-regulated protein kinase (ERK) activation (Friedman et al., 2002). In this study, we have investigated the role of ERK in -AR-mediated vasodilatation. Hearts from pigs of both sexes were obtained from a local abattoir. Ring segments from the distal portion of porcine coronary arteries were dissected and mounted in a 4-channel myograph (Danish Myotechnology Ltd.) containing Krebs-Henseleit solution gassed with O2:CO2 (95:5) at 37°C. Tissues were pre-incubated for 45 min with the inhibitor of ERK activation, PD98059, or vehicle (0.1-0.26% DMSO v/v) and then contracted with either KCl or U46619, a thromboxane receptor agonist, before relaxations were induced using cumulative concentrations of vasodilator agents. Effects of PD98059 were assessed using a paired Student’s t-test with data from at least five separate animals. Isoprenaline (1x10-9 M – 1x10-6 M) induced a concentration-dependent vasodilatation of pre-contracted vessel segments. Pre-incubation with PD98059 (50 M) shifted the concentration-response curve to the left (pEC50 values: control = 7.5 ± 0.1, +PD98059 = 8.1 ± 0.1; P0.05), without significantly altering the maximal response (control 109 ± 3.1; +PD98059 122.3 ± 4.9 % relaxation). Responses to 30 µM salbutamol were also enhanced (control 54 ± 8, +PD98059 92 ± 12 % relaxation; P<0.05), while xamoterol-evoked relaxations were unaltered (pEC50 control = 5.9 ± 0.3, +PD98059 = 6.1 ± 0.1; Rmax control = 57 ± 10, +PD98059 = 61 ± 5 % relaxation). Relaxation evoked by direct activation of adenylyl cyclase using forskolin (1x10-9 M – 1x10-6 M) was also unaffected in the presence of PD98059 (pEC50 control = 7.3 ± 0.1, +PD98059 = 7.4 ± 0.1; Rmax control = 126 ± 6, +PD98059 = 115 ± 4 % relaxation). Similarly, in vessels precontracted with KCl instead of U46619, relaxation responses were insensitive to PD98059. These data indicate that ERK is able to regulate -AR-mediated vasodilatation. Inhibition of ERK activation was associated with an enhanced response, indicating that ERK exerts an inhibitory effect on -AR-mediated relaxation. The effect appears to be receptor subtype-dependent, since responses evoked by the 2-AR-selective agonist salbutamol, but not the 1-AR-selective agonist xamoterol, were sensitive to ERK inhibition. The lack of effect of ERK inhibition on relaxation evoked by forskolin suggests that ERK signals through a route independent of the adenylyl cyclase/cAMP/protein kinase pathway during 2-AR-mediated vasodilatation. In tissues pre-contracted with KCl, MEK inhibition had no effect on responses, suggesting that ERK may regulate -AR vasorelaxation by interacting with K+ channels. Cardillo C et al. (1997) Hypertension 30,918-921. Friedman J et al. (2002) Mol Pharmacol 62, 1094-1102. White R et al. (2001) J Pharmacol Exp Ther 298, 917-924. Bacterial outer membranes: lipopolysaccharide and the role of lipid rafts in host invasion – solid state NMR study. Filip Ciesielski, David Griffin, Michael Rittig and Boyan Bonev School of Biomedical Sciences and the Institute of Infection and Immunity, University of Nottingham A growing number of human intracellular pathogens are found to target cholesterol-rich microdomains (lipid rafts) in the host cell membranes. Lipopolysaccharide (LPS), an integral part of the outer membranes of Gram-negative bacteria and an important virulence factor, has been proposed to play a role in mediating lipid raft-dependent host cell invasion. The ultimate aim of this project is to characterize the interaction of LPS and lipid rafts on the molecular level. High-resolution nuclear magnetic resonance (NMR) spectroscopy from natural abundance 13C in lipid membranes is able to provide information from all parts of the membrane simultaneously. At present, solid state magic angle spinning (MAS) NMR spectroscopy is adapted to study the molecular structure and dynamics of LPS and of phospholipid bilayers as membrane models. Model membranes were studied using 13C-NMR at different temperatures under Lee-Goldburg proton decoupling. J-resolved NMR spectra were obtained and used to create a dynamics model of the individual segments of membrane lipids. Simultaneously, a protocol for full reconstitution of LPS into liposomes is being developed for use in NMR experiments, such as heteronuclear correlation 2D NMR, in order to investigate molecular details of LPS-lipid raft interaction. Also, a computational methodology is being developed for molecular dynamic simulations of lipid bilayer systems and LPS characteristics in eukaryotic membranes. It is expected that this approach will provide a suitable method for investigating complex systems involving LPS and lipid rafts. The effect of hypoxia and oxidative stress on STAT3 regulation in primary cardiomyocytes Emma Evans, Samuel Shelton, Peter Shaw Signal Transducer and Activator of Transcription 3 (STAT3) protein has fundamental importance for vertebrate organisms including roles in cell proliferation, differentiation and survival. STAT3 has also been implicated with normal heart development and function. The activity of STAT3 is induced in cardiomyocytes by Leukaemia Inhibitory Factor (LIF)1, whereas the down regulation of STAT3 was observed in myocardial tissue from patients with dilated cardiomyopathy2. Interestingly STAT3 also appears to be important for hypoxic preconditioning, a process which promotes cardio-protection against subsequent ischemia/reperfusion injury3. Reactive oxygen species (ROS) are induced following hypoxic preconditioning and as STAT3 can be regulated by oxidative stress4 it is feasible that STAT3 activation may also be sensitive to oxygen levels in cells. This project aims to look at STAT3 activity and its contribution to the survival of cardiomyocytes during hypoxia/oxidative stress. A redox-insensitive STAT3 will also be use to see whether it offers greater protection to cardiomyocytes against hypoxia/oxidative stress. Understanding the mechanisms of STAT3 regulation in cardiomyocytes will be important for the development of therapeutic approaches for progressive ischaemic heart disease.
1. Kodama et al, (1997), Leukaemia inhibitory factor, a potent cardiac hypertrophic cytokine, activates the AK/STAT pathway in rat cardiomyocytes. Circ. Res. 81, 656-663. 2. Podewski et al, (2003) Alterations in Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling in patients with end-stage dilated cardiomyopathy. Circulation 107, 798- 802. 3. Smith et al, (2004) Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning. Cardiovasc. Res. 63, 611-616. 4. Simon et al, (1998) Activation of the JAK-STAT pathway by reactive oxygen species. Am. J. Physiol. 275, C1640-1652.
School of Biomedical Sciences, University of Nottingham, NG7 2UH, England. ATP-binding cassette (ABC) transporters constitute a large family of membrane embedded pumps that are widely distributed in almost all organisms and are involved in the transport of a wide variety of substances. Seven main subfamilies of ABC transporters, namely A through G, have been recognized and described in humans based on sequence homology [1-3]. ABCG2, or breast cancer resistance protein (BCRP), is a half transporter with one nucleotide binding domain (NBD) at its N-terminus and one transmembrane domain (TMD) at its C-terminus. It is believed to adopt higher order oligomerisation levels in order to become functional. ABCG2 is most highly expressed in the placenta, followed by the brain, liver, prostate, small intestine, and colon. In cancer cells, it can be over expressed and confer resistance to certain anticancer drugs, such as mitoxantrone, doxorubicin, and daunorubicin, by decreasing their cellular accumulation [4]. In addition, ABCG2 effluxes certain fluorescent drugs which enable the use of assays which detect the functionality of different ABCG2 variants or mutants in different cell lines [5]. This study aims to test the effect of mutating some highly conserved residues on the expression and functionality of ABCG2 in HEK293T cells. Residues for mutation were designed following coevolution and statistical coupling analyses (SCA). Novel mutants were compared to three mutants that have predictable effects on the activity and the glycosylation status of ABCG2. The level of expression and the subcellular localization of the mutants were detected by means of fluorescent and confocal microscopy. The function of mutants was analyzed by extrusion of the fluorescent drug BODIPY-FL-Prazosin. Results from both localization and functional studies are still ongoing. [1] Velamakanni S., Wei SL., Janvilisri T. and van Veen HW. ABCG transporters: structure, substrate specificities and physiological roles (A Brief Overview). J Bioenerg Biomembr (2007) 39:465-471. [2] Unadkt JD and Mao Q. Role of the breast cancer resistance protein (ABCG2) in drug transport. The AAPS Journal (2005); 7 (1) Article 12, pp.E118-E133. [3] Varadi A., Sarkadi B., Köblös G. and özvegy-Laczka C. Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition. Biochimica et Biophysica Acta 1668 (2005) 53-63. [4] H. Kusuhara, Y. Sugiyama, ATP-binding cassette, subfamily G (ABCG family), Pflugers Arch 453 (2007) 735-744. [5] Bates SE. et al. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). Biochimica et Biophysica Acta 1512 (2001) 171-182. Transposition of the human mariner Hsmar1 element Corentin Claeys Bouuaert and Ronald Chalmers School of Biomedical Sciences, University of Nottingham, Nottingham, NG7 2UH, United Kingdom. Transposons are segments of DNA capable of moving from one location to another in their host genome. They are phylogenetically widespread, inducing genomes rearrangements and various types of mutations. Cut-and-paste transposons encode an enzyme, the transposase, that excises the element from the donor substrate and subsequently inserts it into a target site. mariner elements are a particularly widespread family of cut-and-paste transposons and are thought to use a fundamentally different mechanism than other systems studied to date. Indeed, most transposases bring together the two transposon ends before they initiate catalysis (synapsis), and the double strand breaks are generated using a DNA-hairpin intermediate. This intermediate is absent in mariner and several lines of evidence also suggested that mariner is able to act on a single transposon end. We have reconstituted transposition of the Hsmar1 element in vitro using purified components. The intermediates and products of the reactions were analyzed using one and two dimensional gels. By examining the topology of the intramolecular integration products we show that excision results from concerted cleavage at each end of a single transposon segment. Our results suggest that catalysis is dependent of synapsis and, unlike other transposons, synapsis is the rate-limiting step in mariner transposition and happens preferentially by meeting at nodes of supercoiled DNA instead of by random collision. Protein import in the Trichomonas vaginalis hydrogenosome, - a mitochondrially derived organelle. Christopher Kay, Jody Smith & Sabrina Dyall Centre for Biochemistry & Cell Biology, School of Biomedical Sciences, University of Nottingham.
The parasite thrives in microaerophilic environments and exploits novel methods to metabolise in the near absence of oxygen. Most eukaryotes possess mitochondria, organelles that enable aerobic respiration, T.vaginalis has instead a derived organelle called the hydrogenosome. Additionally whilst the mechanisms of protein import seem in passing similar to mitochondria the hydrogenosome lacks homologues to many protein translocation components found in all mitochondria. Thus in this project we attempt to uncover the hydrogenosomal proteins of outer membrane translocase. Antioxidant and cytotoxicity properties of selected indigenous fruits of Borneo Island
1School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, NG7 2UH, United Kingdom 2Laboratory of Natural Products, Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Locked Bag No. 2073, 88999, Kota Kinabalu, Sabah, Malaysia 3Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor, Malaysia Research into the antioxidant and anticancer properties of fruits has increased in recent years. Phytochemicals with antioxidant properties, such as polyphenols, carotenoids and anthocyanins, have been suggested to exert such properties. Mangifera pajang or bambangan (in Malay) is a species of plant in the mango family, found in Borneo. Meanwhile, Artocarpus odoratissimus, also called tarap (in Malay), is a tree related to jackfruit, cempedak and breadfruit, and is native to Borneo. The objective of the present study is to investigate the antioxidant and polyphenol phytochemicals properties of the crude extracts of the kernel, peel and flesh of M. pajang, and the seed and flesh from A. odoratissimus, and to study the cytotoxic mechanism(s) against selected cancer cell lines (liver, breast, colon and ovary). Overall, the antioxidant activity of the crude extracts were in decending order M. pajang kernel > M. pajang peel > A. odoratissimus seed > M. Pajang flesh > A. odoratissimus assessed using DPPH free radical scavenging and ferric reducing activities. For cytotoxicity study, the results showed that the kernel ethanol extract of M pajang induced cytotoxicity in MCF-7 (hormone-dependent breast cancer cell) and MDA-MB-231 (non-hormone dependent breast cancer cell) with IC50 values of 23 and 30.5 µg/ml, respectively. This kernel extract induced cell cycle arrest in MCF-7 cells at Sub-G1 (apoptosis) in a time-dependent manner. Interestingly, for MDA-MB-231, the kernel extract induced strong G2-M arrest in cell cycle progression after 24 hours, resulting in high Sub-G1 (apoptosis) arrest after 48 and 72 hours of incubation. Annexin V-FITC and propidium iodide staining revealed that the apoptosis occurred as early as 36 hours post-incubation for MCF-7 cells and 24 hours for MDA-MB-231 cells. This apoptosis appears to be caspase-2 and caspase-3 dependent in MCF-7 cells and caspase-2, caspase-3 and caspase-9 dependent in MDA-MB-231 cells. These findings suggest the potential of M. pajang kernel extract as a potent cytotoxic agent in both hormone and non-hormone dependent breast cancer cell lines. The mechanisms may involve caspases activation, inhibition of DNA synthesis and G2-M cell cycle arrest leading to the induction of apoptosis. VPA produces deficits in hippocampal dependent cognition and inhibits neurogenesis Jariya Umka, Sarah Mustafa, Maha Elbeltagy, Aaran Thorpe, Liaque Latif, Geoffrey Bennett & Peter Wigmore Abstract Valproic acid (VPA) is widely used clinically, as an anticonvulsant and mood stabilizer but is, however, also known to block cell proliferation through its ability to inhibit histone deacetylase enzymes. There have been a number of reports of cognitive impairments in patients taking VPA. In this investigation we wished to examine the relationship between cognition and changes in cell proliferation within the hippocampus, a brain region where continued formation of new neurons is associated with learning and memory. Treatment of rats by ip injection of VPA, reduced cell proliferation in the neurogenic region of the hippocampus and this was linked to a significant impairment in their ability to perform a hippocampal-dependent behavioral test. In addition, drug treatment caused a reduction in BDNF and Notch1 but not doublecortin levels within the hippocampus. These results support the idea that VPA may cause cognitive impairment and provide a possible mechanism for this by reducing neurogenesis within the hippocampus. Investigating cortical descending control of the peripheral auditory system Darren Edwards and Alan Palmer Institute of Hearing Research, Science Road, University of Nottingham, Nottingham, NG7 2RD Corticofugal projections may modulate auditory signal processing in many subcortical nuclei including the thalamus, inferior colliculus (IC) and superior olivary complex. Originating in the olivary complex the olivocochlear bundle, consisting of the lateral and medial (MOC) systems, extends descending control to the cochlea. Here we investigated the effect of reversible cortical inactivation on the guinea pig cochlea. A cooling technique was used to deactivate the cortex in anaesthetized guinea pigs, while a round window electrode monitored the cochlear microphonic (CM) and cochlear action potential (CAP). Classically, activation of the MOC system leads to an increase in the CM and a decrease in the CAP amplitude, a result replicated in studies either directly stimulating the MOC or indirectly via IC stimulation. During cortical inactivation CM and CAP thresholds were elevated, most prominently in the 3-7 kHz range. The effects on the CM were generally greater than on the CAP. Reactivation of the cortex led to a recovery of both CM and CAP to control levels. A reduction and subsequent recovery in CM and CAP output at suprathreshold levels was also apparent, manifesting itself at various times and frequencies during the study. These results indicate that even in the anaesthetized animal the cortex is exercising some control over the function of the cochlea. Probing the dependence of localisation ability on signal detectability: A methodological study Joseph Sollini, Dr R Mason and Dr C Sumner. MRC (IHR) To ensure that chronic implants in ferrets yield as much data as possible during their operating time it may prove fruitful to create a task which could be used to study a number of different phenomena. Therefore, we wished to know whether a localisation task (approach to target) could also be used to measure detection thresholds. Results have demonstrated that ferrets, like humans, can localise pure tones to a high degree of accuracy when sufficiently separated in space. Furthermore they can do so in the presence of a wideband noise masker. Finally they can perform above chance levels close to their predicted detection thresholds. However a number of refinements will be necessary to improve the data being generated. Bias seems to exist within the ferret performance and so it was necessary to investigate whether this is indeed bias or another factor within the booth. Some of the factors considered were the sound production and room reverberation, these were tested to see the effect this may have on performance. In addition control of reward delivery was investigated to understand its role in the demonstrated behaviour. Finally analyses capable of reducing the effects of bias were investigated. Protective effect of folate and vitamin C for cardiac defects induced by ethanol in chick embryos: the role of Connexins. Memon S. & Pratten.M.K. School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK, NG7 2UH. The teratogenic effects of ethanol are growth retardation, craniofacial abnormalities, malformation of the nervous and cardiovascular systems. Periconceptional supplementation with multivitamins containing folic acid and vitamin C has been shown to help prevent neural tube defects. Various studies also support an association between multivitamins and reduction in the occurrence of congenital heart disease. One possible mechanism for teratogens which cause heart defects is the disruption of gap junction formation, probably by interference with the assembly of connexins. This study was conducted to evaluate the adverse effects of ethanol on chick embryos in ovo and also to examine the protective effects of folic acid and vitamin C. Embryos were injected on day 3 of incubation with 100µl of ethanol (100µl/ml), vitamin C (100µM), folic acid (1mM) or ethanol plus vitamin C or folic acid. Control embryos were treated with PBS only. On day 9 of development embryos were examined for viability, growth retardation and any gross malformations. After gross examinations the hearts were dissected out and processed for either H&E staining or Western blotting for the study of the role of connexin 43, the major connexion involved in fetal heart development. Results showed that ethanol significantly decreased overall survival of embryos. The majority of surviving embryos were growth retarded and grossly malformed (p<0.05). Addition of vitamin C or folic acid to ethanol could significantly improve survival of embryos, and embryos were not growth retarded or malformed (p>0.05). Histological analyses of hearts showed abnormalities in all the 4 chambers of hearts with ethanol treated embryos, while vitamin C and folic acid (in addition to ethanol) treated hearts were comparable to the control. Western blotting showed a reduction in the level of connexin 43 in ethanol treated embryos, which was significantly different to controls and vitamin C and folic acid treated embryos. Supplementation with multivitamins containing vitamin C during pregnancy may be a useful therapy to prevent defects in heart development that may be brought about by ethanol. |