• Amoebae promote persistence of epidemic strains of MRSA.

      Huws, Sharon A.; Smith, Anthony W.; Enright, Mark C.; Wood, Pauline J.; Brown, Michael R. W. (Wiley InterScience, 2006)
      The control of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infection is of concern worldwide. Given the evidence that several pathogenic species replicate within amoebae and emerge more virulent and more resistant and the abundance of amoebae in healthcare settings, we investigated interactions of Acanthamoeba polyphaga with epidemic MRSA isolates. MRSA proliferated in the presence of amoebae, attributable partly to intracellular replication. Following 24 h of co-culture, confocal microscopy revealed that c. 50% amoebae had viable MRSA within phago-lysosomes and 2% of amoebae were heavily infected with viable cocci throughout the cytoplasm. Infection control strategies should recognize the contribution of protozoa.
    • Antimicrobial agents and biofilms

      Brown, Michael R. W.; Smith, Anthony W. (Cambridge University Press, 2003)
      Interest in biofilms has increased dramatically in recent years. New microscopic and molecular techniques have revolutionized our understanding of biofilm structure, composition, organization, and activities. This book brings advances in the prevention and treatment of biofilm-related diseases to the attention of clinicians and medical researchers. Human tissues often support complex microbial communities growing as biofilms that can cause infections. As microbes in biofilms exhibit increased tolerance toward anti-microbial agents and decreased susceptibility to host defense systems, biofilm-associated diseases have become increasingly difficult to treat. (Cambridge University Press)
    • Antioxidants and AP-1 activation: a brief overview.

      Gómez del Arco, Pablo; Martínez-Martínez, Sara; Calvo, Victor; Armesilla, Angel Luis; Redondo, Juan Miguel (Elsevier BV, 1997)
      Activity of the transcription factor AP-1 is controlled by different MAPK cascades that regulate the different AP-1 components at the transcriptional and posttranscriptional level. Recently, AP-1 has been shown to behave as a redox-sensitive transcription factor that can be induced under both pro-oxidative and antioxidative conditions. In this overview we summarize the signaling pathways that converge on the activation of AP-1 and the components of these pathways that have been shown to be targets of antioxidants. The activation of AP-1 by antioxidants may account for the expression of a number of genes that mediate important functions under physiological conditions.
    • Applications of cell-penetrating peptides as signal transduction modulators.

      Jones, Sarah; Howl, John D. (Washington, D.C.: CRC Press, 2006)
      THIS BOOK: Since the first Handbook of Cell-Penetrating Peptides was prepared in 2001, the wealth of new information on the use of these peptides as transport systems has in fact served to confound the field. The constant internal change in the field of cell-penetrating peptides (CPPs) is due to recent research uncovering apparent ambiguities in cellular uptake. There is still neither a common terminology nor a uniform explanation for the penetrative mechanism of cell-penetrating peptides. In this second edition of the Handbook of Cell-Penetrating Peptides, the authors summarize the current state of the field including recent reevaluations of earlier studies of CPP mechanisms. Beginning with an overview of the classes of peptides and their individual uptake mechanisms, from the earlier lipid models to the more recent endocytotic pathways, the book demonstrates the diversity and the opportunity for these biologically active proteins to serve as future drug leads. The text then covers the use of CPPs in gene modulation, addressing the application of antisense and decoy oligonucleotides, as well as the new avenue of research targeting specific tumors and other tissues-questions that had barely been asked when the first edition was published. (CRC Press)
    • Biofilms and protozoa: a ubiquitous health hazard

      Smith, Anthony W.; Brown, Michael R. W. (IWA Publishing, 2003)
      This timely book will introduce its readers to the structure and function of biofilms at a fundamental level as determined during the past decade of research, including: Extracellular polymers as the biofilm matrix; Biofilm phenotype (differential gene expression, interspecies signalling); Biofilm ecology; Biofilm monitoring; Resistance of biofilms to antimicrobial agents and Biofilm abatement. (IWA Publishing)
    • Biofilms, dormancy and resistance

      Smith, Anthony W.; Brown, Michael R. W. (Cambridge University Press, 2003)
      All cellular life-forms can exist in replicating and non-replicating states. Organisms replicate only when the conditions are beneficial, and when not replicating they concentrate on survival of these environmental stresses. Many bacteria, harmful to humans, survive the period of infection in a low growth state. This book addresses the basic science of microbial dormancy and low growth states, putting this in the context of human medicine. Such fundamental topics as bacterial growth and non-growth, culturability and viability are covered, as well as survival of the host’s immune response, and inter-bacterial signalling. Following this introduction, more medically-focused topics are discussed, namely antibiotic resistance arising during stationary phase, biofilms, the bacteria which cause gastric ulcers and tuberculosis as the classic persistent bacterial infection. This book will be of interest to graduate students and researchers in medical microbiology, immunology and infectious disease medicine who are interested in bacterial dormancy in relation to disease. (Cambridge University Press)
    • Biological applications of the receptor mimetic peptide mastoparan.

      Jones, Sarah; Howl, John D. (Betham Science Publishers, 2006)
      The receptor mimetic and mast cell degranulating peptide mastoparan (MP) translocates cell membranes as an amphipathic alpha-helix, a feature that is undoubtedly a major determinant of bioactivity through the activation of heterotrimeric G proteins. Chimeric combinations of MP with G protein-coupled receptor (GPCR) ligands has produced peptides that exhibit biological activities distinct from their composite components. Thus, chimeric peptides such as galparan and M391 differentially modulate GTPase activity, display altered binding affinities for appropriate GPCRs and possess disparate secretory properties. MP and MP-containing chimerae also bind and modulate the activities of various other intracellular protein targets and are valuable tools to manipulate and study enzymatic activity, calcium homeostasis and apoptotic signalling pathways. In addition, charge delocalisation within the hydrophilic face of MP has produced analogues, including [Lys5, Lys8,Aib10]MP, that differentially regulate mast cell secretion and/or cytotoxicity. Finally, the identification of cell penetrant variants of MP chimerae has enabled the effective intracellular delivery of non-permeable biomolecules and presents an opportunity to target novel intracellular therapeutic loci.
    • Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

      Martínez-Martínez, Sara; Gómez del Arco, Pablo; Armesilla, Angel Luis; Aramburu, Jose; Luo, Chun; Rao, Anjana; Redondo, Juan Miguel (American Society for Microbiology, 1997)
      Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants.
    • Bradykinin receptors as a therapeutic target

      Howl, John D.; Payne, Sarah J. (London: Informa Healthcare, 2003)
      Biologically-active kinins, including bradykinin (BK) and Lys(0)-BK (kallidin), are short-lived peptide mediators predominantly generated by the enzymatic action of kallikreins on kininogen precursors. A diverse spectrum of physiological and pathological actions attributed to local kinin production is a consequence of the activation of G-protein-coupled receptors (GPCRs). Currently, two major subtypes of kinin receptor, designated B(1) and B(2), are recognised, although there is much evidence for pharmacological heterogeneity, particularly within the B(2) receptors. Considering these facts and the widespread distribution of kinin receptors in many human tissues, it is no surprise that the therapeutic potential of kinins and kinin receptor antagonists remains the focus of numerous investigations. Studies in animals and animal tissues, instrumental in elucidating the biological roles of kinins, are well-documented in numerous excellent reviews. Unfortunately, and despite the enormous potential illustrated by animal studies, attempts to develop kinin analogues as therapeutic agents to combat human disease have largely proven disappointing. Consequently, this review selectively focuses upon studies that are directly relevant to the targeting of human BK receptors as a therapeutic intervention. In addition to providing a succinct review of well-documented pathological conditions to which kinin receptors contribute, the authors have also included more recent data that illustrate new avenues for the therapeutic application of kinin analogues.
    • Cannabinoid receptor systems: therapeutic targets for tumour intervention.

      Jones, Sarah; Howl, John D. (Taylor & Francis (Informa Healthcare), 2003)
      The past decade has witnessed a rapid expansion of our understanding of the biological roles of cannabinoids and their cognate receptors. It is now certain that Delta9-tetrahydrocannabinol, the principle psychoactive component of the Cannabis sativa plant, binds and activates membrane receptors of the 7-transmembrane domain, G-protein-coupled superfamily. Several putative endocannabinoids have since been identified, including anandamide, 2-arachidonyl glycerol and noladin ether. Synthesis of numerous cannabinomimetics has also greatly expanded the repertoire of cannabinoid receptor ligands with the pharmacodynamic properties of agonists, antagonists and inverse agonists. Collectively, these ligands have proven to be powerful tools both for the molecular characterisation of cannabinoid receptors and the delineation of their intrinsic signalling pathways. Much of our understanding of the signalling mechanisms activated by cannabinoids is derived from studies of receptors expressed by tumour cells; hence, this review provides a succinct summary of the molecular pharmacology of cannabinoid receptors and their roles in tumour cell biology. Moreover, there is now a genuine expectation that the manipulation of cannabinoid receptor systems may have therapeutic potential for a diverse range of human diseases. Thus, this review also summarises the demonstrated antitumour actions of cannabinoids and indicates possible avenues for the future development of cannabinoids as antitumour agents.
    • CEL1: a novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose.

      Armesilla, Angel Luis; Thurston, Christopher F.; Yague, Ernesto (Blackwell Publishing, 1994)
      The cel1 gene of Agaricus bisporus encodes a protein (CEL1) that has an architecture resembling the multi-domain fungal cellulases, although the sequence of its putative catalytic core is not matched by any other in the protein and nucleic acid data bases. The N-terminal half of the putative catalytic domain of CEL1 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase. The fusion protein was used to raise a CEL1-specific antibody. CEL1 was detected as an extracellular 49.8 kDa protein in A. bisporus cellulose-grown cultures, where it bound strongly to cellulose. CEL1 was neither an endoglucanase, a cellobiohydrolase able to hydrolyze fluorogenic cellobiosides, a beta-glucosidase, a xylanase, nor a cellobiose: quinone oxidoreductase. CEL1 was present in some fractions of culture fluid separated by electrophoresis which released soluble sugars from crystalline cellulose.
    • Cell penetrating peptides as signal transduction modulators

      Jones, Sarah; Howl, John D. (CRC Press (Taylor & Francis), 2007)
      THIS BOOK: Since the first Handbook of Cell-Penetrating Peptides was prepared in 2001, the wealth of new information on the use of these peptides as transport systems has in fact served to confound the field. The constant internal change in the field of cell-penetrating peptides (CPPs) is due to recent research uncovering apparent ambiguities in cellular uptake. There is still neither a common terminology nor a uniform explanation for the penetrative mechanism of cell-penetrating peptides. In this second edition of the Handbook of Cell-Penetrating Peptides, the authors summarize the current state of the field including recent reevaluations of earlier studies of CPP mechanisms. Beginning with an overview of the classes of peptides and their individual uptake mechanisms, from the earlier lipid models to the more recent endocytotic pathways, the book demonstrates the diversity and the opportunity for these biologically active proteins to serve as future drug leads. The text then covers the use of CPPs in gene modulation, addressing the application of antisense and decoy oligonucleotides, as well as the new avenue of research targeting specific tumors and other tissues-questions that had barely been asked when the first edition was published. By summarizing the diffuse information regarding CPPs, including the ambiguities and variety of mechanisms, the Handbook of Cell-Penetrating Peptides provides the most solid foundation available from which to expand the potential of this rapidly growing field of medicine. (CRC Press)
    • Charge delocalisation and the design of novel mastoparan analogues: enhanced cytotoxicity and secretory efficacy of [Lys5, Lys8, Aib10]MP.

      Jones, Sarah; Howl, John D. (Elsevier BV, 2004)
      The formation of an amphipathic helix is a major determinant of the biological activity of the tetradecapeptide mastoparan (MP). To address the functional significance of lysyl residues at positions 4, 11 and 12 of MP, we synthesised five novel analogues using sequence permutation and arginine-substitution to delocalise cationic charge. Comparative bioassays determined cytotoxicity, beta-hexoseaminidase secretory efficacy and peptide-activated extracellular receptor-stimulated kinase (ERK)1/2 phosphorylation. The monosubstitution of individual lysine residues with arginine produced differential changes to the indices of cytotoxicity and secretion indicating that these conservative substitutions are compatible with membrane translocation and the selective binding and activation of intracellular proteins. More profound changes to the predicted hydrophilic face of MP, resulting from the relocation or substitution of additional lysyl residues, enhanced both the cytotoxicity and secretory efficacy of novel peptides. Significantly, the more amphipathic peptide [Lys5, Lys8, Aib10]MP was identified to be both the most cytotoxic and the most potent secretagogue of all the peptides compared here. Charge delocalisation within the hydrophilic face of MP analogues was also compatible with peptide-induced activation of ERK1/2 phosphorylation. Our data indicate that charge delocalisation is a suitable strategy to engineer more potent analogues of MP that differentially target intracellular proteins.
    • Chimeric peptides as tumour-selective delivery systems.

      Jones, Sarah; Howl, John D. (Society for Neuro-oncology and Duke University Press, 2005)
      The cell-type-specific targeting of cytotoxic agents and other functional moieties can be achieved by using peptidyl address motifs that selectively bind protein targets expressed at high density at the cell membrane. Indeed, numerous studies have confirmed the utility of ligands for G protein–coupled receptors as components of heterofunctional peptide chimeras that are selective biological probes. Our current efforts are directed toward the further development of chimeric peptidyl constructs that employ sequences derived from GPCR ligands or cell penetrant motifs to affect the selective delivery of cytotoxins and signal transduction modulators to tumor cells. We have designed and synthesized a range of hybrid constructs consisting of cytotoxins (peptide and non-peptide) covalently linked to an address peptide derived from the C-terminal of gastrin (G7; H-AYGWMDF-NH2). The G7 homing motif targets a novel binding site expressed by U373MG astrocytic tumor cells that is distinct from classical CCK1/CCK2 receptors. Moreover, biological responses following activation of this novel membrane-bound protein may offer additional therapeutic advantages. For example, G7 receptor activation is reported to inhibit the motility of malignant astrocytoma in vivo while avoiding the growth-promoting effects of gastrin (Pannequin et al., J. Pharmacol. Exp. Ther. 302, 274, 2002). We evaluated the cytotoxicity of our chimeric peptides by comparing changes in cellular viability using MTT conversion assays. Our data indicate that chimeric peptides dose-dependently and rapidly (<8 h) reduced the viability of U373MG cells. Moreover, as a chimeric amino-terminal extension, the G7 address motif enhanced the cytotoxicity of both mastoparan (H-INLKALAALAKKIL-NH2) and D(KLAKLAK)2 peptides reported to stimulate necrosis and/or apoptosis of eukarytoic cells. In conclusion, hybrid G7 chimeras enhance the efficacy of cytotoxic agents and may be valuable probes to investigate and manipulate additional aspects of astrocytoma cell biology. This work was supported by The Wellcome Trust.
    • Chimerism: a strategy to expand the utility and applications of peptides

      Howl, John D. (Clifton, N.J.: Humana Press, 2005)
      The modular nature of peptides can be exploited in the synthesis of chimeric sequences that combine diverse motifs in a single molecule. A theoretical consideration of the classification of peptides further expounds the multigeneric nature of peptide chimeras. Strategies for chimeric peptide syntheses include the chemical cross-linking of monomers and tandem combination by conventional SPPS. Additional details of chimeric peptide synthesis are also provided elsewhere in this volume. This chapter also explores some of the more common applications of chimeric peptides with particular emphasis on the molecular pharmacology of sequences that include address motifs for G protein-coupled receptors. Specific details of the biological properties of chimeras containing mastoparan, an amphiphilic tetradecapeptide component of wasp venom, further illustrate the novel and often unpredictable biological actions of chimeric constructs. These and numerous additional studies confirm that chimerism is an established strategy for the synthesis of molecular probes and bioactive agents.
    • Critical value determination on similarity of fingerprints

      Fang, Kai-Tai; Liang, Yi-Zeng; Yin, Xiao-lin; Chan, Kelvin C.; Lu, Guang-Hua (Elsevier, 2006)
      A high-performance liquid chromatographic (HPLC) fingerprint of Chinese Angelica (CA) was developed basing on the consistent chromatograms of 40 CA samples (Angelica sinensis (Oliv.) Diels). The unique properties of this HPLC fingerprints were validated by analyzing 13 related herbs including 4 Japanese Angelicae Root samples (JA, A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyame Hikino), 6 Szechwan Lovage Rhizome samples (SL, Ligusticum chuanxiong Hort.) and 3 Cnidium Rhizome samples (CR, Cnidium officinale Makino). Both correlation coefficients of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. The amount of senkyunolide A in CA was less than 30-fold of that in SL and CR samples, which was used as a chemical marker to distinguish them. JA was easily distinguished from CA, SL and CR based on either chromatographic patterns or the amount of coniferyl ferulate. No obvious difference between SL and CR chromatograms except the relative amount of some compounds, suggesting that SL and CR might have very close relationship in terms of chemotaxonomy. Ferulic acid and Z-ligustilide were unequivocally determined whilst senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, butylphthalide, E-ligustilide, E-butylidenephthalide, Z-butylidenephthalide and levistolide A were tentatively identified in chromatograms based on their atmospheric pressure chemical ionization (APCI) MS data and the comparison of their UV spectra with those published in literatures.
    • Design, synthesis and applications of cell-penetrant peptides as signal transduction modulators

      Howl, John D.; Farquhar, Michelle; Jones, Sarah (Springer Verlag, 2006)
      THIS BOOK: Understanding Biology Using Peptides: Proceedings of the 19th American Peptide Symposium highlights many of the recent developments in peptide science, with a particular emphasis on how these advances are being applied to basic problems in biology and medicine. Specific topics covered include novel synthetic strategies, peptides in biological signaling, post-translational modifications of peptides and proteins, peptide quaternary structure in material science and disease, and peptides as tools in drug discovery. (Springer Verlag)
    • Development and validation of the Chinese Quality of Life Instrument.

      Leung, Kwok-fai; Liu, Feng-bin; Zhao, Li; Fang, Ji-qian; Chan, Kelvin C.; Lin, Li-zhu (BioMed Central, 2005)
      BACKGROUND: This paper describes the development of the Chinese Quality of Life Instrument (ChQOL) which is a self-report health status instrument. Chinese Medicine relies very much on asking subjective feelings of patients in the process of diagnosis and monitoring of treatment. For thousands of years, Chinese Medicine practitioners have accumulated a good wealth of experiences in asking questions about health of their patients based on the concept of health in Chinese Medicine. These experiences were then transformed into questions for the ChQOL. It is believed that ChQOL can contribute to the existing Patient Report Outcome measures. This paper outlines the concept of health and disease in Traditional Chinese Medicine, the building of the conceptual framework of the ChQOL, the steps of drafting, selecting and validating the items, and the psychometric properties of the ChQOL. METHODS: The development of the ChQOL was based on the concept of health in Traditional Chinese Medicine with a theory driven approach. Based on the results of literature review, the research team developed an initial model of health which encompassed the concept of health in TCM. An expert panel was then invited to comment and give suggestions for improvement of the initial model. According to their suggestions, the model was refined and a set of initial items for the ChQOL was drafted. The refined model, together with the key domains, facets and initial items of the ChQOL were then mailed to a sample of about 100 Chinese medicine practitioners throughout Mainland China for their comments and advice. A revised set of items were developed for linguistic testing by a convenience sample consisting of both healthy people and people who attended Chinese Medicine treatment. After that, an item pool was developed for field-testing. Field test was conducted on a convenience sample of healthy and patient subjects to determine the construct validity and psychometric properties of the ChQOL. RESULTS: Construct validity was established by various methods, i.e. the internal consistency in all facets and domains were good; the correlation between facets to domain, and domains to overall ChQOL correlation were high; confirmatory factor analysis showed that the structure fitness of all facets, domain and overall structure were good with CFI > 0.9. Test-retest reliability was also good, especially in the domain scores with ICC value ranging from 0.83 to 0.90. No ceiling or floor effect was noted which indicated that ChQOL can be applied to subjects with a wide range of health status. Most facet scores, domain scores and the overall CHQOL scores were able to discriminate groups of subjects with known differences in health status. The ChQOL had mild positive convergence with the other generic health related QOL measures, i.e. the WHOQOL-100 and the SF-36, with moderate correlations. CONCLUSION: In conclusion, the study indicated that the ChQOL is conceptually valid with satisfactory psychometric properties. It can provide additional information on health and QOL on top of the existing generic health related QOL measures. Furthermore, it forms basis for further testing and applications in clinical trials.
    • Development of high-performance liquid chromatographic fingerprints for distinguishing Chinese Angelica from related umbelliferae herbs.

      Lu, Guang-Hua; Chan, Kelvin C.; Liang, Yi-Zeng; Leung, Kelvin Sze-Yin; Chan, Chi-Leung; Jiang, Zhi-Hong; Zhao, Zhong-Zhen (Elsevier, 2005)
      A high-performance liquid chromatographic (HPLC) fingerprint of Chinese Angelica (CA) was developed basing on the consistent chromatograms of 40 CA samples (Angelica sinensis (Oliv.) Diels). The unique properties of this HPLC fingerprints were validated by analyzing 13 related herbs including 4 Japanese Angelicae Root samples (JA, A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyame Hikino), 6 Szechwan Lovage Rhizome samples (SL, Ligusticum chuanxiong Hort.) and 3 Cnidium Rhizome samples (CR, Cnidium officinale Makino). Both correlation coefficients of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. The amount of senkyunolide A in CA was less than 30-fold of that in SL and CR samples, which was used as a chemical marker to distinguish them. JA was easily distinguished from CA, SL and CR based on either chromatographic patterns or the amount of coniferyl ferulate. No obvious difference between SL and CR chromatograms except the relative amount of some compounds, suggesting that SL and CR might have very close relationship in terms of chemotaxonomy. Ferulic acid and Z-ligustilide were unequivocally determined whilst senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, butylphthalide, E-ligustilide, E-butylidenephthalide, Z-butylidenephthalide and levistolide A were tentatively identified in chromatograms based on their atmospheric pressure chemical ionization (APCI) MS data and the comparison of their UV spectra with those published in literatures.