• TWIST1 regulates the activity of ubiquitin proteasome system via the miR-199/214 cluster in human end-stage dilated cardiomyopathy

      Baumgarten, Anna; Bang, Claudia; Tschirner, Anika; Engelmann, Anke; Adams, Volker; von Haehling, Stephan; Doehner, Wolfram; Pregla, Reinhard; Anker, Markus S.; Blecharz, Kinga; et al. (Elsevier, 2013-09-30)
      The transcription factor TWIST1 has been described to regulate the microRNA (miR)-199/214 cluster. Genetic disruption of TWIST1 resulted in a cachectic phenotype and early death of the knock-out mice. This might be connected to the activity of the ubiquitin-proteasome-system (UPS), as miR-199a has been suggested to regulate the ubiquitin E2 ligases Ube2i and Ube2g1.
    • Intervention effects of Ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of Neurotrophin-4 and N-Cadherin

      Wang, Shu-Qiu; Li, Xiao-Jie; Zhou, Shaobo; Sun, Di-Xiang; Wang, Hui; Cheng, Peng-Fei; Ma, Xiao-Ru; Liu, Lei; Liu, Jun-Xing; Wang, Fang-Fang; et al. (PLoS, 2013-04-24)
      Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg2+ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg2+ free medium for 3 hours), iii) GLS group I (incubated with Mg2+ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg2+ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.
    • The effects of methionine acquisition and synthesis on Streptococcus pneumoniae growth and virulence

      Basavanna, Shilpa; Chimalapati, Suneeta; Rubbo, Bruna; Yuste, Jose; Hosie, Arthur H.F.; Thomas, Gavin H.; Brown, Jeremy S.; Maqbool, Abbas; Ogunniyi, Abiodun D.; Paton, James C. (PLoS, 2013-01)
      Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a KD of ~25 nM, and a ΔmetQ strain had reduced uptake of C14-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/ΔmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 – Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.
    • Increased collagen synthesis rate during wound healing in muscle

      Zhou, Shaobo; Salisbury, Jonathan; Preedy, Victor R.; Emery, Peter W.; Roy, Sudipto (Public Library of Science, 2013)
      Wound healing in muscle involves the deposition of collagen, but it is not known whether this is achieved by changes in the synthesis or the degradation of collagen. We have used a reliable flooding dose method to measure collagen synthesis rate in vivo in rat abdominal muscle following a surgical incision. Collagen synthesis rate was increased by 480% and 860% on days 2 and 7 respectively after surgery in the wounded muscle compared with an undamaged area of the same muscle. Collagen content was increased by approximately 100% at both day 2 and day 7. These results demonstrate that collagen deposition during wound healing in muscle is achieved entirely by an increase in the rate of collagen synthesis.
    • G-CSF rescues tumor growth and neo-angiogenesis during liver metastasis under host angiopoietin-2 deficiency

      Im, Jae Hong; Tapmeier, Thomas; Balathasan, Lukxmi; Gal, Annamaria; Yameen, Sabira; Hill, Sally; Smart, Sean; Noterdaeme, Olivier; Kelly, Matthew; Brady, Michael; et al. (Wiley, 2013)
      Suppression of neo-angiogenesis is a clinically used anti-tumor strategy with new targets such as angiopoietin-2 (Ang2) being proposed. However, the functions of Ang2 in vascular remodeling, inflammation and tumor growth are not consistent. We examined effect of depletion of host Ang2 on liver colony formation using Ang2 deficient (Ang22/2) mice.
    • Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia

      Springer, Jochen; Tschirner, Anika; Hartman, Kai; Palus, Sandra; Wirth, Eva K.; Ruis, Silvia Busquets; Möller, Nadine; von Haehling, Stephan; Argiles, Josep M.; Köhrle, Josef; et al. (Wiley, 2012-11-01)
      Cachexia is a common co-morbidity in cancer occurring in up to 80% of patients depending on the type of cancer. Uric acid (UA), the end-product of the purine metabolism, is elevated in cachexia due to tissue wasting and upregulated xanthine oxidase (XO) activity. High serum UA levels indicate increased XO-dependent production of oxygen free radicals (reactive oxygen species; ROS) and correlate with metabolic illness and poor survival. We hypothesized that XO-inhibition might reduce inflammatory signals accounting for tissue wasting and improve survival in experimental cancer cachexia. Animals were inoculated intraperitoneally with AH-130 hepatoma cells and treated with two XO-inhibitors: allopurinol [Allo, low (LD) and high dose (HD) 4 and 40 mg/kg/d] and its more effective active metabolite oxypurinol (Oxy, 4 and 40 mg/kg/d) or placebo for 15 days. Weight loss and tissue wasting of both fat and lean tissue (assessed by NMR-scanning) was reduced by both LD and HD Allo and LD-Oxy, but not by HD-Oxy. A robust induction of XO-activity for generation of reactive oxygen species was seen in the placebo group (assessed by electron paramagnetic spectroscopy), which was reduced by XO-inhibition. Increased ROS induced cytokine signaling, proteolytic activity and tissue degradation were all attenuated by XO inhibition. Survival was significantly and dose dependently improved. Food intake and spontaneous locomotor activity were higher, indicating a higher quality of life. Inhibition of XO can reduce tissue wasting and improve survival in cancer cachexia and clearly clinical studies are needed.
    • Elucidation of xenobiotic metabolism pathways in human skin and human skin models by proteomic profiling

      van Eijl, Sven; Zhu, Zheying; Cupitt, John; Gierula, Magdalena; Götz, Christine; Fritsche, Ellen; Edwards, Robert J.; Rodrigues-Lima, Fernando (Public Library of Science, 2012-07-26)
      Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin.
    • Genetic susceptibility to experimental autoimmune glomerulonephritis in the wistar kyoto rat

      Reynolds, John; Cook, Paul R.; Behmoaras, Jacques; Smith, Jennifer; Bhangal, Gurjeet; Tadros, Susan; Tee, Jonathan; Salama, Alan D.; Evans, David J.; Aitman, Timothy J.; et al. (Elsevier, 2012-05)
      In experimental autoimmune glomerulonephritis (EAG), a model of Goodpasture's disease, Wistar Kyoto (WKY) rats immunized with collagenase-solubilized glomerular basement membrane (GBM) or the recombinant NC1 domain of the α3 chain of type IV collagen [α3(IV)NC1] develop anti-GBM antibodies and focal necrotizing glomerulonephritis with crescent formation. However, Lewis (LEW) rats, which share the same major histocompatibility complex (MHC) haplotype, are resistant to EAG development. A genome-wide linkage analysis of backcrossed animals with EAG revealed a major quantitative trait locus (QTL) on rat chromosome 13 (LOD = 3.9) linked to the percentage of glomerular crescents. To investigate the role of this QTL in EAG induction, reciprocal congenic rats were generated (LEW.WCrgn1 congenic and WKY.LCrgn1 congenic), immunized with recombinant rat α3(IV)NC1, and assessed for EAG development. WKY.LCrgn1 rats showed a marked reduction in albuminuria, severity of crescentic nephritis, and number of glomerular macrophages compared with WKY controls. No reduction in antibody levels was observed. However, LEW.WCrgn1 rats were resistant to EAG development, as were LEW controls. Macrophage activation in vitro was assessed in parental and congenic rat bone marrow-derived macrophages (BMDMs). WKY.LCrgn1 BMDMs showed a significant reduction in Fc receptor-mediated oxidative burst, phagocytosis of opsonised polystyrene beads, and LPS-induced levels of MCP-1 secretion and iNOS mRNA expression compared with WKY rats. These results confirm the importance of Crgn1 on chromosome 13 in EAG susceptibility, mediated partly through differences in Fc receptor-mediated macrophage activation.
    • Recombination adenovirus-mediated human lactoferrin cDNA inhibits the growth of human MCF-7 breast cancer cells.

      Wang, Jianjie; Li, Qingwang; Ou, Yetao; Li, Kun; Han, Zengsheng; Wang, Peijun; Zhou, Shaobo; Yanshan University (Royal Pharmaceutical Society of Great Britain, 2012-03)
      Human lactoferrin, an 80 kDa iron-binding glycoprotein, has antitumour effects. We have explored the potential therapeutic role of re-expressing human lactoferrin gene product in human breast cancer.
    • Increased incidence of anti-GBM disease in Fcgamma receptor 2b deficient mice, but not mice with conditional deletion of Fcgr2b on either B cells or myeloid cells alone.

      Sharp, Phoebe E.H.; Martin-Ramirez, Javier; Boross, Peter; Mangsbo, Sara M.; Reynolds, John; Moss, Jill; Pusey, Charles D.; Cook, H. Terence; Tarzi, Ruth M.; Verbeek, J. Sjef; et al. (Elsevier, 2012-02)
      Fcgamma receptor 2b (Fcgr2b) is the only inhibitory Fcgamma receptor in both humans and mice, and is implicated in both antibody production and effector responses to antibody complexes. Reduced function of Fcgr2b has previously been associated with anti-glomerular basement membrane antibody (anti-GBM) disease in mice. However, the mice used had 129 genetic elements flanking the deleted Fcgr2b gene, which are known to increase susceptibility to autoimmunity. In order to confirm a role for Fcgr2b in protection from anti-GBM disease, wild type (WT) mice, mice lacking Fcgr2b on a pure C57BL/6 background, or mice lacking Fcgr2b on a C57BL/6 background with 129 flanking sequences, were immunized with the recombinant NC1 domain of alpha 3 Type IV collagen. Twenty two weeks after immunization, there was a higher incidence of crescentic glomerulonephritis, macrophage infiltration and renal dysfunction in both groups of Fcgr2b-/- mice, indicating an important role of Fcgr2b in regulating the development of anti-GBM disease, on both genetic backgrounds. In order to determine the cellular origin of the Fcgr2b-associated effect, disease was induced in mice with deficiency of Fcgr2b on either B cells alone (CD19Cre), or a subset of myeloid cells (LysozymeMCre). Neither B cell nor myeloid specific knockout mice developed crescentic glomeruonephritis with higher incidence than WT mice indicating that Fcgr2b deficiency on either B cells or a subset of myeloid cells alone is not sufficient to increase susceptibility to anti-GBM disease, but that a combination of cell types, or deficiency of Fcgr2b in a different cell type, is also required.
    • Stimulation of the PD-1/PDL-1 T-cell co-inhibitory pathway is effective in treatment of experimental autoimmune glomerulonephritis

      Reynolds, John; Sando, Gregg S.; Marsh, Olivia B.; Salama, Alan D.; Evans, David J.; Cook, H. Terence; Pusey, Charles D.; Imperial College London (Oxford University Press, 2012)
    • Caspase-2 and caspase-8 trigger caspase-3 activation following 6-OHDA-induced stress in human dopaminergic neurons differentiated from ReNVM stem cells

      Chaudhry, Zahara Latif; Ahmed, Bushra Y. (Maney Publishing, 2012)
      The purpose of the study was to establish a suitable model to study Parkinson’s disease (PD) pathogenesis in differentiated dopaminergic neurons (dDCN). The specific aim was to demonstrate the involvement of the caspase family and to identify specific caspases which are activated by 6-hydroxydopamine (6OHD) treatment leading to death of dDCN.
    • Development and validation of a standardized protocol to monitor human dietary exposure by metabolite fingerprinting of urine samples

      Favé, Gaëlle; Beckmann, Manfred; Lloyd, Amanda J.; Zhou, Shaobo; Harold, Graham; Lin, Wanchang; Tailliart, Kathleen; Xie, Long; Draper, John; Mathers, John C. (Springer, 2011-12)
    • Role of symbiotic auxotrophy in the rhizobium-legume symbioses

      Prell, Jurgen; Bourdès, Alexandre; Kumar, Shalini; Lodwig, Emma M.; Hosie, Arthur H.F.; Kinghorn, Seonag; White, James P.; Poole, Philip S.; Rutherford, Julian (PLoS, 2011-11-10)
      Symbiotic auxotrophy occurs in both determinate pea and indeterminate bean nodules demonstrating its importance for bacteroid formation and nodule function in legumes with different developmental programmes. However, only small quantities of branched chain amino acids are needed and symbiotic auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants inoculated on different legumes probably reflects altered timing of amino acid availability, development of symbiotic auxotrophy and nodule developmental programmes.
    • Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments.

      Zampolla, Tiziana; Spikings, Emma; Srirangarajah, Srimathuri; Rawson, David M.; Zhang, Tiantian; University of Bedfordshire (Cryo Letters, 2011-11)
      Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution.
    • Binding modes of diketo-acid inhibitors of HIV-1 integrase: a comparative molecular dynamics simulation study.

      Huang, Meilan; Grant, Guy H.; Richards, W. Graham; Queens University Belfast (Elsevier, 2011-06)
      HIV-1 integrase (IN) has become an attractive target since drug resistance against HIV-1 reverse transcriptase (RT) and protease (PR) has appeared. Diketo acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN: however the action mechanism is not well understood. Here, to study the inhibition mechanism of DKAs we performed 10 ns comparative molecular dynamics simulations on HIV-1 IN bound with three most representative DKA inhibitors: Shionogi inhibitor, S-1360 and two Merck inhibitors L-731,988 and L-708,906. Our simulations show that the acidic part of S-1360 formed salt bridge and cation-π interactions with Lys159. In addition, the catalytic Glu152 in S-1360 was pushed away from the active site to form an ion-pair interaction with Arg199. The Merck inhibitors can maintain either one or both of these ion-pair interaction features. The difference in potencies of the DKA inhibitors is thus attributed to the different binding modes at the catalytic site. Such structural information at atomic level, not only demonstrates the action modes of DKA inhibitors but also provides a novel starting point for structural-based design of HIV-1 IN inhibitors.
    • Strain differences and the genetic basis of experimental autoimmune anti-glomerular basement membrane glomerulonephritis.

      Reynolds, John; University of Bedfordshire (Blackwell Publishing, 2011-06)
      Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis, caused by autoimmunity to a component of the GBM, the non-collagenous domain of the α3 chain of type IV collagen [α3(IV)NC1]. To investigate the mechanisms of inflammation in glomerulonephritis and to test new approaches to treatment, animal models of glomerulonephritis, termed experimental autoimmune glomerulonephritis (EAG), have been developed in susceptible strains of rats and mice. This review article describes how these models of EAG have been developed over the past three decades, discusses the evidence for the involvement of both humoral and cell-mediated immunity in the induction and pathogenesis of glomerulonephritis in these models and highlights recent, emerging data that have identified potential candidate genes that may control the genetic susceptibility in these different strains of rats and mice. The identification of these susceptibility genes has lead to a better understanding of the genetic basis of this model of anti-GBM disease, which may be relevant to the immunopathogenesis of Goodpasture's disease, and more generally to the progression from autoimmunity to target-organ damage.
    • Requirement for class II phosphoinositide 3-kinase C2  in maintenance of glomerular structure and function

      Harris, D.P.; Vogel, P.; Wims, M.; Moberg, K.; Humphries, J.; Jhaver, K.G.; DaCosta, C.M.; Shadoan, M.K.; Xu, N.; Hansen, G.M.; et al. (American Society for Microbiology, 2011-01)
    • Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (Junceella juncea and Junceella fragilis) to cryoprotectants.

      Tsai, Sujune; Spikings, Emma; Huang, I-C.; Lin, C.; Mingdao University (Cryo Letters, 2011-01)
      Coral reefs provide a valuable habitat for many economically valuable fish and invertebrates. However, they are in serious jeopardy, threatened by increasing over-exploitation, pollution, habitat destruction, disease and global climate change. Here, we examined the effect of cryoprotectant exposure on mitochondrial activity and membrane potential in coral oocytes in order to find suitable cryoprotectants towards their successful cryopreservation. According to the No Observed Effect Concentrations (NOECs), methanol was found to be the least toxic cryoprotectant whilst DMSO was the most toxic cryoprotectant. The results also demonstrated that there were no significant differences (p > 0.05) in ATP concentrations between Junceella juncea and Junceella fragilis after exposure to all concentrations of all cryoprotectants for 30 min. Using confocal microscopy, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) staining indicated that the mitochondrial membrane potential of Junceella fragilis oocytes reduced after 1 M and 2 M methanol treatment and a loss of the mitochondrial distribution pattern and poor green fluorescence after 3M methanol treatment. Therefore, even oocytes that show no adverse effect of cryoprotectants on survival might suffer some more subtle impacts. The results obtained from this study will provide a basis for development of protocols to cryopreserve the oocytes of gorgonian corals.